The phorbol ester PMA efficiently inhibits the in vitro IFN-alpha and -beta responses in human blood monocytes induced by Sendai virus and in natural IFN-producing cells induced by glutaraldehyde-fixed herpes simplex virus-infected human amnion (WISH) cells. This PMA-mediated inhibition of IFN-alpha/beta secretion is correlated with considerably reduced levels of IFN-alpha/beta mRNA, as determined by Northern blot analysis. Nuclear run-on assays further show that, at least in monocytes, PMA inhibits transcription of the IFN-alpha and -beta genes. The synthetic diacylglycerol 1-oleoyl-2-acetylglycerol has the same effects as PMA, and the protein kinase inhibitor staurosporine prevents the PMA-mediated inhibition of IFN-alpha/beta expression. These results suggest a role for protein kinase C in the inhibition of IFN-alpha/beta responses. The PMA does not inhibit the accumulation of IFN-beta mRNA in monocytes stimulated by Sendai virus in the presence of cycloheximide, indicating that the inhibitory action of the phorbol ester requires de novo protein synthesis.