Human plasma and serum levels for lα, 25-dihydroxyvitamin D were determined by a cytosol radioreceptor assay (RRA) and a radioimmunoassay (RIA). For both assays, 1.5 ml of human serum or plasma is used. Prior to RRA or RIA, extraction with benzene is performed followed by ‘high-performance’ liquid chromatography (HPLC) on a silica column (25 × 0.46 cm) with hexane isopropanol (9/1 by vol), to isolate 1α,25-dihydroxyvitamin D from the other vitamin D metabolites. The cytosol receptor was isolated from the intestine of healthy chickens. The antisera were raised in rabbits to 1α,25-dihydroxyvitamin D 3-3-hemisuccinate coupled to bovine serum albumin. The standard curves for RRA and RIA are prepared with 1α,25-dihydroxyvitamin D 3. 1α,25-dihydroxy[ 3H]vitamin D 3 of high spec act (158 kCi/mol) is used as tracer. The reactants are incubated for 16 h at 4°C. Then, bound and free ligand are separated after the addition of dextran-coated charcoal. Both assays have a sensitivity of 2 pg/tube. The cytosol receptor and the antibodies have about the same absolute affinity for 1α,25-dihydroxyvitamin D 3 but the cytosol receptor has a higher relative affinity for 1α,25-dihydroxyvitamin D 3 (compared with other vitamin D metabolites). Reproducibility and precision are better for the RIA. The between-and within-assay CVs are 16.0% ( x = 58.7 ng/l , n = 16) and 11.2% ( x = 52.1 ng/l , n = 15), respectively, for RRA and 12.6% ( x = 61.8 ng/l , n = 27) and 7.4% ( x = 61.8 ng/l , n = 15), respectively using RIA. Reference values obtained by both assays on healthy males and healthy premenopausal females are the same for both sexes; 53.9 ±31.0 ng/l ( n = 46) using RRA and 51.8 ± 30.2 ng/l ( n = 91) for RIA (mean ± 2 sd).