We report here the first radioimmunoassay for a vitamin D metabolite utilizing a radioiodinated tracer. Antibodies were generated in a goat immunized with the vitamin D analog 23, 24, 25, 26, 27-pentanor-C(22)-carboxylic acid of vitamin D, coupled directly with bovine serum albumin. The 125I-labeled tracer was prepared by reacting a 3-amino-propyl derivative of vitamin D-C(22)-amide with Bolton-Hunter reagent. The primary antiserum, used at a 15,000-fold final dilution, cross-reacted equally with all cholecalciferol and ergocalciferol metabolites tested except 1,25-dihydroxycalciferol metabolites and the parent calciferols; the antiserum did not cross-react with dihydrotachysterol. Calibrators were prepared in vitamin D-stripped human serum. 25-Hydroxycholecalciferol was quantitatively extracted from serum or plasma (50 microL) with acetonitrile. The assay consists of a 90-min incubation at room temperature with primary antiserum, followed by a 20-min incubation with a second antiserum and separation of bound from free fractions by centrifugation. The detection limit of the assay was 2.8 micrograms/L for 25-hydroxycholecalciferol. Results with the present assay compared well with those from a liquid-chromatographic procedure involving specific ultraviolet detection of 25-hydroxycalciferol in plasma.