Granulosa cells obtained from immature estradiol-treated SD rats (10 rats/experiment) were employed in elucidating the control mechanism of steroid secretion. Phorbol 12-myristate 13-acetate (PMA) inhibited estradiol production by cultured rat granulosa cells with IC50 less than 1 nM. PMA, however, stimulated small but significant increases in progesterone production in a dose-dependent manner with ED50 of 14 nM to 3.5-fold above the basal control level. These effects could not be induced by calcium ionophore A23187. Forskolin-stimulated progesterone production was inhibited by the concomitant addition of PMA with IC50 less than 1 nM. The phosphorylation of proteins by [32P] orthophosphate-labelled cells was examined by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Treatment of cells with forskolin altered the intensity of 40 kDa acidic phosphoprotein compared to that of the control. On the other hand, treatment of cells with PMA altered the intensity of 78 and 32 kDa acidic phosphoproteins. These results suggest, therefore, that PMA can modulate steroidogenesis in rat granulosa cells, presumably through activation of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C).