A sensitive radioimmunoassay (RIA) for 1 alpha,25-dihydroxyvitamin D [1 alpha,25(OH)2-D] with a double antibody (DAB) separation technique to separate free from bound antigen has been developed. The hormone was extracted from 1 ml serum or plasma by Extrelut columns and normal phase high performance liquid chromatography and quantitated in the DAB-RIA. The detection limit of the assay was 3.75 ng/l. The intraassay variation coefficients were 15.9% and 10.5% for samples with 1 alpha,25(OH)2D3 concentrations of 54 ng/l and 130 ng/l, respectively. The interassay variation coefficients were 18.0% and 16.7% for these two concentrations. Mean (and SD) values for 1,25(OH)2D in serum of 40 healthy subjects and 38 patients with chronic renal failure who did not receive 1,25(OH)2D3 were 62.8 ng/ml (22.2) and 12.4 ng/ml (9.8), respectively. The mean value for 7 patients with primary hyperparathyroidism was 66.5 ng/ml (35.8) before surgery. These results compared well with those of an established charcoal-based RIA. Compared to charcoal-based RIAs, the DAB-RIA is faster and requires less laborious assay procedures.