A proinflammatory cytokine IFN-γ stimulates microglia in the injured brain; however, signaling pathways for IFN-γ-mediated microglia activation are not well characterized. In the present study, a protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) acts in concert with IFN-γ to enhance nitric oxide (NO) production in murine microglial BV2 cells by synergistically increasing expression of inducible NO synthase (iNOS). The synergistic NO production by PMA was in part decreased by a PKC inhibitor Gö6976. PMA alone induced activation of nuclear factor-kappa B (NF-κB) and extracellular signal-regulated kinase (ERK) of mitogen-activated protein kinases (MAPKs) subtypes, whereas IFN-γ alone had little effect. PMA and IFN-γ synergistically enhanced activity of NF-κB, but not ERK. The inhibitors of NF-κB (pyrrolidine dithiocarbamate, PDTC) and ERK (1,4-diamino-2,3-dicyano-1,4 bis[2-aminophenylthio]butadiene; U0126) markedly decreased synergistic NO production in BV2 cells treated with IFN-γ and PMA in combination. We found further that co-treatment with IFN-γ and PMA synergistically induced interferon regulatory factor-1 (IRF-1), which is the major transcription factor for IFN-γ-mediated iNOS expression. The present results demonstrate the cooperative interaction of multiple signaling pathways in the induction of NO production in activated microglial cells, and suggest that the functional interplay of these pathways may be important for the onset of microglia-mediated inflammatory responses in brain. © 2003 Wiley-Liss, Inc.