Production of nitric oxide (NO) by macrophages is important for the killing of intracellular infectious agents. Interferon (IFN)- γ and lipopolysaccharide stimulate NO production by transcriptionally up-regulating the inducible NO synthase (iNOS). Macrophages from mice with a targeted disruption of the IFN regulatory factor-1 (IRF-1) gene (IRF-1 ^-/- mice) produced little or no NO and synthesized barely detectable iNOS messenger RNA in response to stimulation. Two adjacent IRF-1 response elements were identified in the iNOS promoter. Infection with Mycobacterium bovis (BCG) was more severe in IRF-1 ^-/- mice than in wild-type mice. Thus, IRF-1 is essential for iNOS activation in murine macrophages.