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.