The direct effects of estradiol-17 beta (E2), diethylstilbestrol (DES) and tamoxifen on testicular testosterone production by purified immature pig Leydig cells in vitro were studied. Leydig cells were obtained from 3-4 weeks old piglet testes by enzymatical dispersion followed by discontinuous Percoll gradient centrifugation. Leydig cells were treated with E2, DES, and tamoxifen in the absence or presence of LH after 12 h of incubation. Media were collected 48 h later for testosterone and cAMP measurement. E2 did not affect basal testosterone production. When Leydig cells were incubated with increasing concentrations (0.001-10.0 micrograms/ml) of E2, DES, or tamoxifen for 48 h, LH-stimulated testosterone production was reduced. The degree of this reduction was dependent on E2, DES, and tamoxifen, and a concentration of E2 and DES and tamoxifen higher than 100 ng/ml and 10 ng/ml was needed, respectively. DES and tamoxifen also reduced LH-stimulated cAMP formation. When equal concentrations of DES and tamoxifen were added concomitantly to Leydig cells, the inhibition was additive, indicating that tamoxifen does not prevent the inhibitory effects of DES. Forskolin, an activator of adenylate cyclase, stimulated testosterone production to an extent comparable to that attained with LH, and DES and tamoxifen reduced forskolin-stimulated testosterone production. DES and tamoxifen suppressed the conversion of exogenous pregnenolone and progesterone to testosterone, but did not affect the conversion of 17 alpha-hydroxyprogesterone to testosterone, suggesting a specific inhibition of 17 alpha-hydroxylase. These results suggest that E2, DES, and tamoxifen directly inhibit immature pig Leydig cell steroidogenesis, at least in part via an inhibition of cAMP formation and a decrease in the activity of 17 alpha-hydroxylase.