The effect of phorbol ester on the synthesis of nitric oxide (NO) in murine microglial cells was examined. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, alone had no effect, whereas PMA with recombinant interferon (rIFN)-γ synergistically increased NO synthesis in murine microglial cells. The maximal effect of PMA on NO synthesis increase always fitted with the range for full activation of PKC in these cells. The increase of NO synthesis was reflected as increased amount of immunologic NO synthase (iNOS) mRNA detected by Northern blotting. Treatment with PKC inhibitors such as staurosporine or polymyxin B decreased rlFN-γ-plus-PMA-stimuIated NO synthesis. Further, prolonged incubation of the cells with PMA, which down-regulates PKC activity, abolished the synergistic cooperative effect with IFN-γ. NG-monomethyl-L-arginine monohydrate, an analogue of L-arginine, and arginase inhibited rIFN-γ-plus-PMA-induced NO production in murine microglial cells. On the basis of these observations, we conclude that PKC might not be involved in the expression of iNOS, but instead, might be involved in the posttranscriptional modification of iNOS m