The objective of this study was to investigate the effects of 4 beta-phorbol 12-myristate 13-acetate (4 beta-PMA)--a potent activator of protein kinase C--on the responsiveness of mouse Leydig cells to stimulation with rat atriopeptin II (rAP-II). We report that, in these cells, the stimulation of testosterone production by rAP-II could be inhibited in a dose-dependent manner by 4 beta-PMA (1-200 nM). In contrast, the basal steroidogenesis was stimulated 2-fold by 4 beta-PMA. There was no inhibition of testosterone production when the cells were stimulated with 8-bromo cyclic GMP (8Br-cGMP) in the presence of 4 beta-PMA. Furthermore, addition of 4 beta-PMA resulted in a marked reduction in the amount of cGMP accumulated in response to rAP-II stimulation. 4 alpha-Phorbol 12-myristate 13-acetate (4 alpha-PMA) was found to have no effect at all. The inhibitory effect of 4 beta-PMA on steroidogenesis could be completely reversed by the addition of 0.25 mM 3-isobutyl 1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Also, the 4 beta-PMA-induced lowering of cGMP content could be partially reversed by IBMX. Membrane fractions from cells treated with 4 beta-PMA or 4 alpha-PMA did not differ in their contents of either basal or rAP-II-stimulated guanylate cyclase activities. We conclude that the 4 beta-PMA-mediated inhibition of testosterone production by Leydig cells stimulated with rAP-II results from an activation of a phosphodiesterase enzyme, hypothetically through an activated protein kinase C. This leads to a reduction in the cellular cGMP content through an increased metabolic removal of cGMP formed in response to rAP-II stimulation.