The production of nitric oxide by macrophages has been implicated as a host defense mechanism against microbial pathogens and tumor cells. Recent reports have implicated interferon-alpha/beta (IFN-alpha/beta) as an autocrine/paracrine signal critical for the induction of murine iNOS. In this report we have systematically investigated the role of IFN-beta in the induction of iNOS in the murine macrophage cell line, RAW 264.7. First, we demonstrate that IFN-beta expression is highly up-regulated, and is secreted in response to lipopolysaccharide (LPS). Treatment of RAW macrophages with LPS results in a time-dependent phosphorylation of STAT-1 on both tyrosine residue 701 (Tyr-701) and serine residue 727 (Ser-727) that is consistent with the timing of endogenous IFN-beta expression. LPS also induces interferon regulatory factor-1 expression with similar kinetics. We further demonstrate that exogenous IFN-beta accelerates the induction of iNOS by LPS. The acceleration of iNOS induction is observed at the levels of transcription, protein expression, and NO formation. Accordingly, we propose that the cytokine environment of macrophages may determine the rate and magnitude of nitric oxide production, thereby regulating the cytotoxic response to pathogen challenge.