Human embryo fibroblasts (HEF) were primed when treated with a synthetic diacylglycerol, OAG, or the phorbol esters TPA or DBP. These primed HEF produce more interferon-beta (IFN-beta) in response to poly(rI).poly(rC), or poly(rA).poly(rU), added 1 h or 18 h later. These priming agents are activators of protein kinase C (PKC). A PKC inhibitor, H-7, blocked their priming effects and also those of human IFN-alpha. Two phorbol esters, 4PDD and 4P, that did not activate PKC did not prime HEF cells. Pretreatment of HEF cells for 1 h or 18 h with TPA or DBP reduced their susceptibility to infection with vesicular stomatitis virus (VSV); this effect was blocked by treatment with H-7. In contrast, the antiviral effects of IFN-alpha were not blocked by H-7, or by previous down-regulation of PKC by prolonged treatment of HEF cells with TPA. These results show that in HEF cells treated with IFN-alpha PKC plays a role in the processes that prime for IFN production, but not in those which establish the antiviral state.