The actions of clomiphene, tamoxifen, and 17 beta-estradiol were studied in an isolated avian granulosa cell system during short term incubations. A dose-dependent inhibition of ovine LH-enhanced progesterone production was observed for all three compounds, with an IC50 of 3 X 10(-6) M and maximal inhibition (95%) at 5 X 10(-5) M. A similar inhibition was found when cells were stimulated by either 8-bromo-cAMP or forskolin. Neither clomiphene nor tamoxifen had an inhibitory effect on the conversion of pregnenolone to progesterone, a step that was blocked by 17 beta-estradiol and isoxazole , a known inhibitor of 3 beta-hydroxysteroid dehydrogenase. On the other hand, cells exposed to clomiphene failed to use [6-3H]25-hydroxycholesterol, while estradiol significantly increased the conversion of labeled substrate to pregnenolone at the expense of progesterone (30% vs. 7% of the total radioactivity). By comparison, in control cells, progesterone represented the major metabolite with 36% of the total radioactivity; pregnenolone had only 2.8%. The results demonstrate that these triphenylethylene compounds, which are used clinically as antiestrogens, inhibit steroidogenesis at a step before pregnenolone formation, probably at the site of the cholesterol side-chain cleavage enzyme complex, while 17 beta-estradiol inhibits 3 beta-hydroxysteroid dehydrogenase.