Bacterial lipopolysaccharide (LPS) induces interferon (IFN) secretion and an antiviral state in murine peritoneal macrophages (PM). These cells secrete predominantly IFN-beta, as shown by neutralization assays with monoclonal antibodies. Secretion of IFN-beta is also induced in PM by IFN-gamma. LPS and IFN-gamma synergistically stimulated PM to produce IFN in amounts almost comparable to those induced by infection with Newcastle disease virus. Low levels of IFN-beta mRNA can be detected in freshly harvested PM by hybridization assays. The accumulation of this mRNA is markedly increased in PM treated with LPS or IFN-gamma, and it is further enhanced in the presence of the inhibitor of protein synthesis, cycloheximide. Similar studies were carried out on the RAW 264.7 line of transformed macrophages. These cells are induced to secrete IFN-beta by LPS but not by IFN-gamma, suggesting that this cytokine may elicit such specific response only in PM. IFN-beta mRNA is undetectable in untreated RAW 264.7 cells, and accumulation of this mRNA is induced by LPS but not by IFN-gamma. The secretion of IFN induced by these agents in PM and by LPS in RAW 264.7 cells and the corresponding accumulation of IFN-beta mRNA are blocked by an inhibitor of protein kinase C, staurosporine. The activity of this kinase is apparently necessary to stimulate accumulation of IFN-beta mRNA. The induction of IFN-beta by IFN-gamma appears to be a characteristic response of PM and may be at least in part responsible for the resistance of these cells to viral infections.