By use of a single-cartridge system, 1,25-dihydroxyvitamin D [1,25(OH)2D] is extracted and purified from a plasma or serum sample. Proteins are removed and 1,25(OH)2D is liberated from the sample with acetonitrile. The acetonitrile extract is applied to the nonpolar octadecylsilanol silica cartridge, in which 1,25(OH)2D is retained while polar compounds are eluted. Then by "phase-switching" on the same cartridge, 1,25(OH)2D is sufficiently resolved from other vitamin D metabolites and extraneous lipophilic compounds to allow its quantification by radioreceptor assay according to an established procedure. Mean (and SD) values for 1,25(OH)2D in serum of 29 normal, 27 chronic renal failure, and nine pregnant subjects were 28.2 (11.3), 10.9 (5.2), and 47.3 (12.9) ng/L, respectively. Results compared well with those of an established radioreceptor procedure. This procedure offers the advantage of a single rapid purification step not involving "high-performance" liquid chromatography or evaporation, under nitrogen, of polar solvents such as acetonitrile or methanol.