Since 25(OH)D is a prohormone it needs to be converted into its active form 1,25-dihydroxy vitamin D (1,25(OH)2D). The conversion of 25(OH)D into 1,25(OH)2D is catalyzed by an enzyme named 1α-hydroxylase (synonym CYP27B1), which is expressed in the kidney and other organs, such as bowel, prostate, breast, pancreas (pancreatic islets), and lungs. Under normal circumstances the majority of circulating 1,25(OH)2D is synthesized in the kidneys. While renal 1,25(OH)2D synthesis is tightly regulated by serum levels of calcium, PTH, and 1,25(OH)2D itself there are other, mainly local factors that regulate 1,25(OH)2D synthesis in extra renal tissues. As a consequence in patients with hypercalcemia, renal impairment and other conditions circulating 1,25(OH)2D levels do not longer reflect the organism’s vitamin D supply. Measurement of 1,25(OH)2D is only required in individuals with specific medical conditions, where 25(OH)D is no longer a reliable surrogate of the organism’s supply with 1,25(OH)2D. Such conditions are renal impairment, sarcoidosis, tuberculosis, 1α-hydroxylase deficiency, and a defective vitamin D receptor (VDR) causing vitamin D resistance.