Vitamin D: Optimal Testing Recommendations

Also known as: 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D

  • 25-hydroxyvitamin D should only be ordered for individuals at risk of vitamin D deficiency and not as a population screening test.
  • Conditions at increased risk: Breastfed infants, older adults, those with limited sun exposure, dark skin, inflammatory bowel disease and other malabsorption disorders, obesity or gastric bypass
  • 1,25-dihydroxyvitamin D should not be ordered to evaluate vitamin D status.
  • 1,25-dihydroxyvitamin D should only be ordered to monitor certain conditions (acquired and inherited disorders of vitamin D metabolism).
  • There are rarely any indications for ordering both 25-hydroxyvitamin D and 1,25-dihydroxyvitamin.

Guidelines for Test Utilization

Can I order this test for pediatric patients?

25-hydroxyvitamin D should only be ordered if vitamin D deficiency is suspected or the patient is at risk of vitamin D deficiency.

1,25-dihydroxyvitamin D should only be ordered in cases where acquired or inherited disorders of vitamin D metabolism are suspected. [back to top]

What does the test tell me?

Vitamin D undergoes several steps of metabolic activation – specifically, hepatic production of 25-hydroxyvitamin D followed by renal production of 1,25-dihydroxyvitamin D. The 25-hydroxyvitamin D represents the dominant form of vitamin D in circulation while 1,25-dihydroxyvitamin represents the active form of vitamin D.

Serum 25-hydroxyvitamin D can be used to screen for vitamin D deficiency in individuals at risk of vitamin D deficiency as it is reflective of vitamin D stores. However, it is not recommended to use 25-hydroxyvitamin D as a population screening tool for vitamin D deficiency.

There are few circumstances where 1,25-dihydroxyvitamin D would be helpful. Serum levels of 1,25-dihydroxhyvitamin D have little to no correlation to vitamin D stores as it is regulated primarily by parathyroid hormone and calcium levels. Unless a disorder (inherited or acquired) of vitamin D metabolism is suspected, 1,25-dihydroxyvitamin D should not be ordered. [back to top]

How should I interpret the results?

Decision limits recommended by national guidelines such as the Institute of Medicine or the Endocrine Society should be used when interpreting Vitamin D test results. Reference ranges should not be used for interpretation of Vitamin D results.

The Endocrine Society recommends a decision limit (optimum level/sufficient level) of 30 – 100 ng/mL (deficient <20, insufficient 21-29 ng/mL), while the Institute of Medicine (IOM) recommends a decision limit (optimum level) of 20-50 ng/mL for the general healthy population. The IOM further clarifies this range by stating that values in the range of 20-50 ng/mL may be insufficient in certain patient populations and that there is no known benefit to values > 50 ng/mL. A vitamin D level of 80 ng/mL is the lowest reported level associated with toxicity in patients without primary hyperthyroidism who have normal renal function. Most patients with toxicity have levels > 150 ng/mL.

For interpretation of 1,25-dihydroxyvitamin D test results, please refer to the following publications on adult or pediatric reference ranges.

When should I order this test? How often?

Routine vitamin D testing is not warranted in the general population (not at risk of deficiency).

25-hydroxyvitamin D can be used for infrequent assessment of vitamin D levels when there is suspicion of deficiency or excess amounts.

Common causes of low 25-hydroxyvitamin D

  • darker skin pigmentation
  • obesity
  • malabsorption syndromes
  • lack of supplementation for breast-fed infants
  • drugs affecting absorption and/or metabolism of vitamin D

Common causes of high 25-hydroxyvitamin D

  • vitamin D toxicity

1,25-dihydroxyvitamin D is limited when there is suspicion of vitamin D metabolism disorder. The frequency of testing will depend on its purpose (diagnosis versus monitoring) and should be determined based on patient needs.

Common causes of low 1,25-dihydroxyvitamin D

  • chronic renal failure
  • hypoparathyroidism

Common causes of high 1,25-dihydroxyvitamin D

  • hyperparathyroidism
  • granulomatous diseases (sarcoidosis)
  • malignancies (1-alpha-hydroxylase-producing tumors) [back to top]

When should I NOT order this test?

Do not order 25-hydroxyvitamin D if there are no indications or symptoms of vitamin D deficiency. The use of 25-hydroxyvitamin D for population screening of vitamin D deficiency may cause misclassification of patients and thus, unnecessary treatment.

Do not order 1,25-dihydroxyvitamin D to assess vitamin D status as serum levels have little to no relationship with body vitamin D stores. The use of 1,25-dihydroxyvitamin D is strictly limited to instances of abnormal vitamin D or phosphate metabolism. [back to top]

Is the test result absolute confirmatory of the condition indicated above? If not, what other tests should I consider?

YES: 25-hydroxyvitamin D levels can confirm vitamin D status to direct further clinical management.

VARIABLE: 1,25-dihydroxyvitamin D levels can confirm level of 1-alpha-hydroxylase activity, but this may require additional tests depending on the disorder suspected. [back to top]

Are there factors that can affect the lab result?

Immunoassay-based measurements of vitamin D may be affected by heterophilic antibodies and/or human anti-mouse antibodies. [back to top]

What follow-up test(s) might be helpful?

Work up for vitamin D deficiency and related disorders may benefit from tests for: Calcium, Phosphate, Parathyroid hormone. [back to top]

Known consequences due to mis-utilizing this test (if applicable)

There is insufficient evidence to assess the benefits and harms of screening for vitamin D deficiency in asymptomatic adults. No clear advantages of screening and early intervention have been demonstrated. [back to top]


Institute of Medicine Committee to Review Dietary Reference Intakes for Vitamin D.

Calcium. The National Academies Collection: reports funded by National Institute of Health. In: Ross AC, Taylor CL, Yaktine AL, Del Valle HB, editors. Dietary reference intakes for calcium and vitamin D. Washington (DC): National Academies Press (US) National Academy of Sciences; 2011.

Holick MF, Binkley NC, Bischoff-Ferrari HA, et al. Evaluation, Treatment, and Prevention of Vitamin D Deficiency: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab, 96(7):1911-1930, 2011.

Ross AC, Manson JE, Abrams SA, Aloia JF, Brannon PM, Clinton SK, Durazo-Arvizu RA, Gallagher JC, Gallo RL, Jones G, Kovacs CS, Mayne ST, Rosen CJ, Shapses SA. The 2011 report on dietary reference intakes for calcium and vitamin D from the Institute of Medicine: what clinicians need to know. J Clin Endocrinol Metab. 2011 Jan;96(1):53-8.

Bouillon R, Rosen C. The IOM – Endocrine Society Controversy on Recommended Vitamin D Targets: In Support of the IOM Position. Vitamin D, Volume 1: Biochemistry, Physiology and Diagnostics, Fourth Edition, 2018. (Chapter 57A – available online, indexed)

Last reviewed: March 2023. The content for Optimal Testing: the Association for Diagnostics & Laboratory Medicine’s (ADLM) Guide to Lab Test Utilization has been developed and approved by the Academy of Diagnostics & Laboratory Medicine and ADLM’s Science and Practice Core Committee.

As the fields of laboratory medicine and diagnostic testing continue to grow at an incredible rate, the knowledge and expertise of clinical laboratory professionals is essential to ensure that patients receive the highest quality and most useful laboratory tests. ADLM’s Academy and Science and Practice Core Committee have developed a test utilization resource focusing on commonly misused tests in hospitals and clinics. Improper test utilization can result in poor patient outcomes and waste in the healthcare system. This important resource geared toward medical professionals recommends better tests and diagnostic practices. Always consult your laboratory director to make sure these recommendations are appropriate for your patient population.