A high-performance liquid chromatographic (HPLC) method for the assay of the hepatic microsomal polysubstrate monooxygenase catalyzed hydroxylation of testosterone is described. The metabolites are extracted from the incubation mixture with dichloromethane and the extract is washed with dilute alkali and water, dried over anhydrous sodium sulfate, and evaporated to dryness. The residue is dissolved in methanol and an aliquot analyzed. The products are separated by reverse-phase chromatography with a methanol/water/tetrahydrofuran gradient and quantitated at 240 nm by the internal standard technique. The assay does not use radioactively labeled testosterone and can measure hydroxylase activity in microsomal samples containing less than 1.0 mg protein. At least seven products, 2 alpha-, 2 beta-, 6 beta-, 7 alpha-, 16 alpha-, and 16 beta-hydroxytestosterone and androstenedione, are resolved by HPLC. The major products formed by microsomes from untreated adult male rats are 2 alpha- (not 2 beta-) and 16 alpha-hydroxytestosterone and androstenedione which constituted 60% of the total products, followed by 6 beta-, 7 alpha-, and smaller quantities of 2 beta- and 16 beta-hydroxytestosterone. The carrier of the substrate in the incubation mixture was found to affect significantly the metabolite pattern and total activity, and of the several solvents studied methanol yielded the highest total activity. Since the 6 beta-, 7 alpha-, and 16 alpha-hydroxylation of testosterone is catalyzed by distinct forms of cytochrome P-450, this assay which measures seven products may serve as a useful qualitative probe of the cytochrome P-450 population of the monooxygenase.