A sensitive protein binding assay for vitamin D is described. The vitamin D3 was extracted from plasma with diethyl ether and methylene chloride. The lipid extract was purified in Sephadex LH-20 followed by Lipidex 5000 and finally by high pressure liquid chromatography on a Zorbax Sil column (0.79 x 25 cm) developed in 0.25:99.75 isopropanol: methylene chloride. The vitamin D fraction was collected and quantitated by competitive protein binding assay with a 1/50,000 dilution of sheep plasma in 0.05 M potassium phosphate buffer (pH 7.5) containing 0.01% gelatin. [H3]-25-Hydroxyvitamin D3 was used as a radioactive tracer in the assay. We found that under these conditions, sheep plasma had equal affinity for vitamin D2 and vitamin D3 and could detect as little as 0.1 ng of vitamin D. When rat, cow, or human plasma was substituted for the sheep plasma, the decline in sensitivity to vitamin D2 was fivefold to tenfold. With this assay, we found excellent agreement (r = 0.98) between the results obtained by competitive protein binding analysis and direct U.V. absorbance analysis by high pressure liquid chromatography.