Group B streptococcus (GBS) is the leading cause of sepsis in neonates. Nitric oxide (NO) release plays a role in the hypotension that characterizes septic shock. It has been shown that GBS beta-hemolysin/cytolysin (beta-h/c) stimulates the transcription of inducible NO synthase (iNOS) in murine macrophages via intracellular pathways similar to those that mediate lipopolysaccharide-induced iNOS activation. Here, it is demonstrated that the GBS cell wall and beta-h/c act synergistically to induce iNOS in interferon (IFN)-gamma-primed [corrected] RAW 264.7 murine macrophages. In nonprimed macrophages, combined activation by the GBS cell wall plus beta-h/c is necessary to induce an NO response, which indicates that both virulence factors cooperate to substitute for the priming signal typically provided by IFN-gamma [corrected].