Superantigens including staphylococcal enterotoxins (SE) bind to major histocompatibility complex class II molecules and interact with T cells bearing particular V beta chains. SEB was shown to induce the expression of interleukin (IL)-1 beta and tumor necrosis factor (TNF)-alpha genes in human peripheral blood monocytes bearing HLA class II molecules. Monoclonal antibodies directed against HLA-DR and -DQ abolished the SEB-induced expression of both the IL-1 beta and TNF-alpha genes, suggesting that the HLA class II molecules mediated the gene expression. Therefore, we investigated the signal transduction mechanism responsible for the expression of IL-1 beta and TNF-alpha genes induced by binding of SEB to the HLA class II molecules. Three protein tyrosine kinase (PTK) inhibitors, genistein, herbimycin A, and tyrphostin, each of which has a different mechanism of action, strongly inhibited the expression of the monokine mRNA induced by SEB. Analyses of PTK activity revealed that SEB induced a rapid increase of membrane-associated PTK activity and this was blocked by tyrphostin. Furthermore, H-7 inhibited the expression of the monokine mRNA induced by SEB, suggesting the involvement of protein kinase C (PKC) in the signaling pathway. The involvement of PKC was confirmed by the observations that phorbol 12-myristate 13-acetate (PMA), a direct activator of PKC, induced the expression of the monokine mRNA and that SEB evoked the activation of membrane-associated PKC. Both activation of PKC and expression of the monokine mRNA induced by SEB appeared to be inhibited by tyrphostin, but those induced by PMA were not. Taken together, these findings indicate that both PTK and PKC play essential roles in HLA class II molecule-mediated signal transduction elicited by SEB and that PTK activation may precede PKC activation in the signaling pathway.