Protein kinase-C (PKC) has been suggested as a possible mediator of the antigonadotropic action of prostaglandin F2 alpha (PGF2 alpha) in luteal cells. To examine this possibility, we evaluated the effects of phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] in relation to those of PGF2 alpha on cAMP accumulation and ATP levels as well as on the subcellular distribution of PKC activity in rat luteal cell cultures. Treatment of luteal cells for 1 h with TPA or PGF2 alpha produced a dose-dependent inhibition of LH-stimulated cAMP accumulation. Maximal inhibition produced by PGF2 alpha was about 35% greater than that produced by TPA. Moreover, PGF2 alpha produced a further inhibition of LH action when the cells were maximally inhibited by TPA. Staurosporine, a PKC inhibitor, reversed inhibition of LH-dependent cAMP accumulation produced by TPA, but had no effect on the response to PGF2 alpha. Furthermore, cells in which PKC was persistently activated by prolonged TPA treatment lost their responsiveness to additional TPA, but continued to show inhibition of cAMP accumulation by PGF2 alpha. TPA also produced a dose-dependent decrease in cell levels of ATP in contrast to PGF2 alpha. Finally, TPA produced a rapid redistribution of PKC activity from the cytosolic to the particulate fraction, whereas PGF2 alpha produced only a slight redistribution. We conclude that the acute antigonadotropic action of PGF2 alpha in rat luteal cells occurs via mechanisms other than phorbol ester-sensitive PKC activation.