IFN-γ induces NO production, inducible NO synthase (iNOS) protein, and promoter expression in mouse macrophage cells. Mutation of IFN regulatory factor 1 responsive element, γ-activated site, as well as NF-κB elements in the murine iNOS promoter strongly reduced IFN-γ-induced iNOS transcriptional activity. The role of NF-κB activation in iNOS induction by IFN-γ was corroborated by overexpression of the NF-κB inhibitory protein IκBα, which inhibited iNOS promoter activity induced by IFN-γ. In addition, IFN-γ treatment induced p65 binding to the iNOS promoter by chromatin immunoprecipitation asay and NF-κB binding to DNA by EMSA, although with a delayed kinetics, suggesting an indirect autocrine role for another cytokine produced in response to IFN-γ. It is interesting that we found that IFN-γ induced TNF-α secretion, and the induction of iNOS expression by IFN-γ was abolished in primary peritoneal macrophages from TNF-α-deficient (TNF-α^−/−) mice or in RAW 264.7 cells treated with anti-TNF-α neutralizing antibodies. Moreover, exogenous addition of recombinant mouse TNF-α restored iNOS expression induced by IFN-γ in TNF-α^−/− mice. It is intriguing that NF-κB binding to DNA in response to IFN-γ treatment was absent in TNF-α^−/− mice. Taken together, our data suggest that the TNF-α produced in response to IFN-γ is required for iNOS induction by activating NF-κB transcription factor.