Tumor necrosis factor alpha (TNF) increased progesterone production in preovulatory rat follicles in vitro. More than 1 h in the presence of TNF was needed to enhance progesterone secretion, which was only seen after 24 h of culture. Neither cAMP nor cGMP levels in media and follicles increased either at short (5-20 min) or long periods (6-24 h) after TNF stimulation. The protein kinase C (PKC) inhibitor, H-7, blocked TNF-stimulated progesterone in a dose-dependent manner (1-300 mM), with 50% inhibition corresponding to 5.2 microM H-7, it also blocked LH-stimulated progesterone production, but higher doses were needed (50% inhibition corresponding to 54.5 microM H-7). However, the cAMP- and cGMP-dependent protein kinase inhibitor, HA1004, did not block TNF stimulated progesterone. The PKC activator, phorbol 12-myristate 13-acetate (PMA), increased progesterone maximally at 32 nM and above. Low doses of PMA in combination with TNF increased progesterone levels above that stimulated by PMA alone; however with the highest does of PMA (320 nM), TNF was unable to increase follicular progesterone secretion. The time course of progesterone stimulation by PMA was similar to that of TNF. H-7 also blocked PMA and PMA + TNF stimulated progesterone accumulation, with a 50% inhibition corresponding to 4.2 and 4.1 microM H-7, respectively. These results indicate that PKC may be a mediator of TNF-stimulated progesterone secretion in preovulatory rat follicles.