Measurement of 5 alpha-reductase activity usually involves quantitation of the radiolabelled products of [3H]testosterone. Recently, however, it has been claimed that the activity of 5 alpha-reductase is masked by the activities of 17 beta-hydroxysteroid dehydrogenase and 3-ketosteroid reductase. Therefore in determining 5 alpha-reductase activity in Hep-G2 cells, we have monitored the concentration of androstenedione to ensure that the conditions for measurement of optimum enzyme activity are maintained. Using a polar (cyano) bonded-phase column and hexane-isopropanol (9:1, v/v) as eluent, the ratio of relative retention times (methyl lithocholate used as the reference standard) of the closest peaks, dihydrotestosterone and estradiol, was 1.2, whilst the highest inter-assay coefficient of variation was 2.7%. Therefore this technique appears suitable for the evaluation of 5 alpha-reductase in cell and tissue samples.