A radioimmunoassay for 1,25-dihydroxycholecalciferol in human serum was described. Antisera to 1,25-dihydroxycholecalciferol-3-hemisuccinate coupled to bovine serum albumin was raised in rabbits and sensitive, high-titer antibodies were obtained. 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/l reduced this interference to 5 and 4%, respectively. 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 chromatographyed on a column of silicic acid. The radioimmunoassay is sensitive to 5 pg/tube (3 ng/l of serum). The between-assay CV [coefficient of variation] 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.