The activities of 3 beta-hydroxysteroid dehydrogenase, 17-hydroxylase, 21-hydroxylase, 11 beta-hydroxylase, C17,20-lyase, and dehydroepiandrosterone sulfotransferase were measured in cultured human fetal definitive zone adrenocortical cells with and without prior exposure to 1 microM ACTH for 48 h. Enzyme induction and measurements of activity were performed using serum- and lipoprotein-free conditions. ACTH induced increases of 5- to 100-fold in the activity of all of these enzymes. Although 3 beta-hydroxysteroid dehydrogenase activity was increased 15-fold, its activity was still an order of magnitude less than that of the hydroxylases. In contrast, when similar experiments were performed using bovine adrenocortical cells, 3 beta-hydroxysteroid dehydrogenase activity was similar to that of the hydroxylases after induction with ACTH. The lower activity of 3 beta-hydroxysteroid dehydrogenase in human cells compared to that in bovine cells resulted in different sequences of transformation of [3H]pregnenolone. The initial product in human cells, before or after induction with ACTH, was 17-hydroxypregnenolone, which was then converted about equally to cortisol (via 17-hydroxyprogesterone and 11-deoxycortisol) and dehydroepiandrosterone sulfate (via dehydroepiandrosterone). In contrast, bovine cells converted pregnenolone to progesterone, with or without prior exposure to ACTH, which was then converted to 17-hydroxyprogesterone, with minimal formation of dehydroepiandrosterone. Adrenal androgen synthesis by human adrenocortical cells thus results from low 3 beta-hydroxysteroid dehydrogenase, which is an intrinsic cell property. Since these experiments were performed using serum-free conditions, cells were not exposed to hormones other than ACTH. The results support the hypothesis that human adrenal androgen synthesis does not require a special hormone.