A radioimmunoassay for the binding protein for vitamin D and its metabolites (DBP) has been developed. Suitable rabbit anti-DBP antiserum was elicited after primary and one booster injection. Anti-DBP antisera, as well as antigroup-specific component antisera, produced a single, monospecific line of percipitation when reacted against purified DBP and human serum. DBP was iodinated with 125I and 125I-DBP was purified by gel filtration on Sephadex G-200. Binding of 125I-DBP by 20 nl of rabbit anti-DBP antisera was approximately 50% and was sharply competed for by 0.4-4.0 ng of DBP standard. Displacement of 125I-DBP by human serum dilutions or standard DBP gave identical curves, and only weak competition was observed with old and new world primate sera. Apo- and holo-DBP possessed indistinguishable immunoreactivity. The assay detects DBP in 1-10 nl of human serum with reasonable accuracy and with reasonable intra- and interassay precision. The mean serum concentration (+/- SEM) for a group of 40 normal adults was 525 +/- 24 mug/ml and no sex difference was observed. Higher levels were found in sera from pregnant women and women receiving oral contraceptives, and decreased concentrations were observed in premature cord and hypoproteinemic sera. No significant correlation between serum DBP levels and serum 25-hydroxycalciferol levels was found, and the DBP content of sera from vitamin D-deprived and vitamin D-treated subjects was indistinguishable from that of normal adults. DBP accounts for 6- of the alpha globulin in normal human serum. Considering the normal serum content of the parent vitamin and its metabolites to be approximately 0.1-0.2 mum, these immunoassay data confirm previous saturation analyses of human serum antiricketic sterol-binding capacity and suggest that greater than 95% of DBP circulates as the apoprotein under normal conditions.