Many practitioners become confused when ordering a vitamin D test. Because 1,25-dihydroxyvitamin D is the active form of vitamin D, many practitioners think that measuring 1,25-dihydroxyvitamin D is an accurate means to estimate vitamin D stores and test for vitamin D deficiency, which is incorrect. Current Endocrine Society guidelines recommend screening for vitamin D deficiency in individuals at risk for deficiency. Serum levels of 1,25-dihyroxyvitamin D have little or no relationship to vitamin D stores but rather are regulated primarily by parathyroid hormone levels, which in turn are regulated by calcium and/or vitamin D. In vitamin D deficiency, 1,25-dihydroxyvitamin D levels go up, not down. Unregulated production of 1,25-dihydroxyvitamin D (i.e., sarcoidosis, granulomatous diseases) is an uncommon cause of hypercalcemia; this should be suspected if blood calcium levels are high and parathyroid hormone levels are low and confirmed by measurement of 1,25-dihydroxyvitamin D. The enzyme that activates vitamin D is produced in the kidney, so blood levels of 1,25-dihydroxyvitamin D are sometimes of interest in patients on dialysis or with end-stage kidney disease. There are few other circumstances, if any, where 1,25-dihydroxyvitamin D testing would be helpful. Serum 25-hydroxyvitamin D levels may be overused, but when trying to assess vitamin D stores or diagnose vitamin D deficiency (or toxicity), 25-hydroxyvitamin D is the correct test.