We investigated the regulation of C3 production by human cultured epidermal keratinocytes by enzyme-linked immunosorbent assay. The results showed that IFNγ and TNFα enhanced the synthesis of C3 by epidermal keratinocytes in a concentration-dependent manner. Moreover, a protein kinase C (PKC) inhibitor blocked C3 production, whereas PMA enhanced it. There was a synergistic effect between IFNγ and TNFα. In experiments to investigate the role of protein tyrosine kinase (PTK) in C3 production, we found that treatment with herbimycin A, a specific inhibitor for the c-Src-related PTK, caused significant enhancement of the C3 production induced by IFNγ or TNFα suggesting that c-Src-type PTK(s) provides a negative signal to C3 production. Each competitive inhibitor of PTK, genistein or tyr-phostin, substantially increased the C3 production by IFNγ at lower concentrations, although each agent had little effect on TNFα-associated production of C3 at the same concentrations. The data show that pro-inflammatory cytokines IFNγ and TNFα synergistically augment C3 production by epidermal keratinocytes by different pathways.