We describe a radioimmunoassay for 1,25-dihydroxycholecalciferol in human serum. We raised antisera in rabbits to 1,25-dihydroxycholecalciferol-3-hemisuccinate coupled to bovine serum albumin, and obtained sensitive, high-titer antibodies. These antibodies had a high affinity for 1,25-dihydroxycholecalciferol and cross reacted mainly with 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol. Addition of 1 mL of normal rabbit serum per liter reduced this interference to 5 and 4%, respectively. However, these interfering steroids are present in large excess, so extensive purification of 1,25-dihydroxycholecalciferol from serum is necessary. The steroid was extracted with ethyl acetate/cyclohexane, purified on Sephadex LH-20, and then chromatographed on a column of silicic acid. The radioimmunoassay is sensitive to 5 pg/tube (3 ng/L of serum). The between-assay CV was 14%. The mean concentration of 1,25-dihydroxycholecalciferol in the serum of 54 healthy adults was 38 (SD 12) ng/L, with no sex-related difference. The assay was further validated by the finding of low or undetectable concentrations in patients with chronic renal failure and of increased concentrations in the serum of patients with primary hyperparathyroidism. In comparison with previously described methods, the major advantage of the present assay is the use of stable gamma-globulins, which are available in large amounts, as binding protein.