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The product of the c-raf-1 proto-oncogene is a cytoplasmic serine/threonine protein kinase that appears to be activated in signal transduction from a variety of cell-surface receptors. The mechanism of c-Raf activation upon stimulation of cell-surface receptors is not clear, but there seem to exist multiple pathways of activation which involve tyrosine and/or serine phosphorylation of the c-Raf protein in vivo. The activated state of Raf is reflected in an increased apparent molecular weight of the Raf protein in sodium dodecyl sulfate-polyacrylamide gels owing to hyperphosphorylation. The tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) is one of the agents able to induce this hyperphosphorylation of Raf in vivo, suggesting that protein kinase C (PKC) may be involved in the activation of c-Raf in particular situations. Using recombinant baculoviruses expressing PKC and Raf polypeptides, we show here that conventional PKC types (alpha, beta, gamma) but not novel types (delta, zeta, eta) or the unrelated Mos kinase are able to activate c-Raf in a TPA-dependent manner upon coexpression in insect cells. Direct phosphorylation of the Raf protein with PKC in vitro also enhanced the kinase activity of c-Raf, suggesting that c-Raf acts immediately downstream of PKC in a protein kinase cascade which is triggered by TPA and may lead to transcriptional activation of TPA-inducible genes and tumor promotion.