Bovine plasma 25-hydroxycholecalciferol was estimated by rapid protein binding assay. Serum-binding proteins in the assay were from rats deficient in vitamin D and from normal rats; no differences existed in standard curves. Nanograms 25-hydroxycholecalciferol in eight cows averaged 45.5 +/- 12.2 ml of plasma. Results from the rapid protein binding assay compared favorably (greater than 10% deviation) with values from a 25-hydroxycholecalciferol assay involving high pressure liquid chromatography.