A new, highly sensitive radioreceptor assay, which does not require high-performance liquid chromatography, has been developed for the determination of 1,25-dihydroxyvitamin D 3 (1,25-(OH 2)D 3) in serum. The assay involves rapid extraction of serum, Sep Pak silica purification, and addition of 1,25-dihydroxyvitamin D 3 receptor, radiolabeled 1,25-dihydroxyvitamin D 3, bovine serum albumin, and monoclonal antibody to specifically precipitate the receptor. This method is sensitive to 0.3–0.6 pg/tube, with B 50 occurring at 5.8 pg/tube. This sensitivity combined with overall recovery of 1,25-dihydroxyvitamin D 3 (81.5 ± 5.2%, n = 50, mean ± SD) allows the measurement of serum 1,25-(OH) 2D 3 in duplicates with only 0.5 ml of serum. Intra- and interassay coefficient of variation were 9.5 and 14.6%, respectively. Dilution analysis, analytical recovery of added 1,25-dihydroxyvitamin D 3, and comparison with a standard method using HPLC have been used to validate the assay. Serum 1,25-dihydroxyvitamin D 3 level was for normal adults, 36.6 ± 10.5 pg/ml ( n = 14); in primary hyperparathyroidism, 98.9 ± 19.9 pg/ml ( n = 16); in chronic renal failure, 17.8 ± 5.1 pg/ml ( n = 12). This method allows large numbers of samples to be processed at once. Further, the method is rapid and provides an accurate assay using small amounts of serum.