Inhibin and activin are gonadal glycoproteins that selectively inhibit and stimulate FSH release, respectively. Previously we have reported that transforming growth factor-beta inhibited hCG-stimulated testosterone formation in mature Leydig cells. In the present study we evaluated the effects of other members of the transforming growth factor-beta family, inhibin and activin, on Leydig cell function. We found that activin (0.1-10 ng/ml) had no effect on basal testosterone formation, but inhibited hCG-stimulated testosterone formation in a dose-dependent manner. Activin (10 ng/ml) inhibited hCG-stimulated testosterone formation by 42%. Activin also inhibited hCG-stimulated cAMP formation. In the presence of activin (5 ng/ml), forskolin (10 microM)- and 8-bromo-cAMP (0.1 mM)-induced testosterone formation were reduced about one third. Conversions of pregnenolone and progesterone to testosterone were also blocked by activin. Interestingly, [125I]hCG binding to Leydig cells and forskolin-induced cAMP formation were not affected by the addition of activin. In contrast to activin, inhibin (0.1-10 ng/ml) had no effect on hCG-induced testosterone formation at any concentration used. However, the inhibitory effects of activin on Leydig cell function were reversed by the concomitant addition of inhibin. Our results suggest that activin inhibits testosterone formation by the Leydig cells derived from normal mature rats. Multiple steps of the steroidogenic pathway are affected by testosterone. Inhibin alone has no effect, but reverses the inhibitory action of activin.