This report describes a microassay for 1,25-dihydroxyvitamin D [1,25-(OH)2D] in plasma which does not require high performance liquid chromatography. The assay involves rapid extraction and preliminary purification on a C-18 Sep-Pak cartridge, followed by final purification on a silica Sep-Pak using hexane-isopropanol. Quantitation of 1,25-(OH)2D is achieved using a nonequilibrium assay employing 1,25-(OH)2D receptor from calf thymus. The method is sensitive to 1.5 pg/tube, with B50 occurring at 9 pg/tube and a useful assay range of 1.5-40 pg/tube. The intra- and interassay coefficients of variation are 6.5% and 11.5%, respectively, and the method is linear over a wide range of sample dilutions. In addition, this assay measures both 1,25-(OH)2D2 and 1,25-(OH)2D3 with equal affinity. The importance of using an assay with equal affinity for 1,25-(OH)2D2 and 1,25-(OH)2D3 is demonstrated by the findings that 25-hydroxyvitamin D2 (250HD2) constituted 38.9% of the total 25-OHD found in clinical samples (12.6 +/- 0.7 ng/ml 25-OHD2 vs. 20.1 +/- 0.5 ng/ml 25-OHD3; n = 807). Results of this new assay have been compared to those of the assay of Horst et al. (21), which employs Sephadex LH-20 and high performance liquid chromatography sample purification. The correlation coefficient was r2 = 0.96, and the slope was 1.05. Using this new assay, plasma 1,25-(OH)2D concentrations were as follows: normal adults, 37.4 +/- 2.2 pg/ml (n = 22); chronic renal failure, 10.6 +/- 1.5 pg/ml (n = 7); anephrics, undetectable (n = 10); infant cord blood, 22.9 +/- 4.4 pg/ml (n = 7); and hyperparathyroidism, 68.9 +/- 5.0 pg/ml (n = 13). This assay should be particularly useful in pediatric or other studies in which sample size is limited.