A B S T R A C T A competitive protein binding assay for measurement of the plasma concentration of la,25-dihydroxyvitamin D3 [la,aD3] has been extended to include the immediate precursor of this hormone, 25-hydroxyvitamin Ds (25-OHD3). In addition, the assay system is capable of measuring the two metabolic products of ergocalciferol, namely, 25-hydroxyvitamin D2 (25-OHD2) and la,25-dihydroxyvitamin D2 [la,25-(OH) 2D2]. The target tissue assay system consists of a high affinity cytosol receptor protein that binds the vitamin D metabolites and a limited number of acceptor sites on the nuclear chromatin. By utilizing a series of chromatographic purification steps, a single plasma sample can be assayed for any of the four vitamin D metabolites either individually or combined. Therefore, the assay procedure allows for both the quantitative and qualitative assessment of the total active vitamin D level in a given plasma sample.To 25-OHD2 as substrate. Comparison of this sterol with la,25-(OH) 2D3 in the assay system showed very similar binding curves; the Da form was slightly less efficient (77%). Comparison of the respective 25-hydroxy forms (25-OHD2 vs. 25-OHD3) at concentrations 500-fold that of la,25-(OH)2D3, again suggested that the binding of the D2 metabolite was slightly less efficient (71%).Finally, the assay was employed to measure the total active vitamin D metabolite pools in the plasma of normal subjects and patients with varying degrees of hypervitaminosis D. The normal plasma levels of 25-OHD and la,25-(OH) 2D measured in Tucson adults were 25-40 ng/ml and 2.1-4.5 ng/100 ml, respectively. Both sterols were predominately (> 90%) in the form of vitamin D3 metabolites in this environment.