The mechanism of gene expression for the MHC I-A beta and TNF genes was studied in murine bone marrow macrophages. The treatment of macrophages with PMA stimulated the expression of TNF, but not I-A beta, suggesting that the TNF gene is responsive to activators of protein kinase C whereas the I-A beta gene is not. The treatment of macrophages with IFN-gamma led to an increase in the level of RNA for both TNF and I-A beta. The increase in expression of I-A beta and TNF, induced by IFN-gamma, was blocked by naphthalenesulfonamide or phenothiazine (trifluoperazine) but was not affected by the addition of isoquinolinesulfonamide or sphingosine. These results suggest that the induced expression of I-A beta and TNF by IFN-gamma is mediated by a pathway that is protein kinase C independent. This was supported by the finding that calcium ionophores were also able to induce the gene expression of both TNF and I-A beta. We observed that when both IFN-gamma and PMA were added to the macrophages, the level of RNA for TNF increased to a higher level than the level seen when either agent alone was added to the cells. In contrast, the addition of both IFN-gamma and PMA to macrophages had an inhibitory effect on the expression of the I-A beta gene. These results further emphasize the complex nature of gene regulation during the activation of macrophages.