Abstract We have previously demonstrated that a 33 kDa C-terminal fragment of c-Raf-1 underwent a mobility shift in response to hydrogen peroxide (H 2O 2) and phorbol myristate acetate (PMA), respectively. In this study, we have demonstrated that H 2O 2 induced the activation of N-terminal deletion mutant as well as full length Raf-1 kinase. The pharmacological PKC activator PMA also induced a weak increase in Raf-1 kinase activity through PKC-ε activation as determined by the transient expression of dominant negative mutants of PKC-ε-K436R. Interestingly, H 2O 2 produced synergistic increase of PMA-stimulated Raf-1 kinase activation after simultaneous treatment of PMA and H 2O 2. This synergistic activation of Raf-1 kinase was further enhanced by cypermethrin (an inhibitor of protein phosphatase 2B) and dephostatin (tyrosine kinase inhibitor) implying an inhibitory role for these phosphatases in the Raf-1 signaling pathway. Taken together, our data suggest that the synergistic activation of Raf-1 kinase in response to PMA and H 2O 2 occurs via mechanisms that involve an interaction of Raf-1 kinase and PKC-ε, along with a transient phosphorylation of both Raf-1 kinase and PKC.