NO is a labile radical involved in several immunological, antimicrobial and inflammatory processes. In macrophages, NO formation is catalyzed by the cytokine-inducible enzyme inducible NO synthase (iNOS). The importance of IFN regulatory factor (IRF)-1 and of the signal transducers and activators of transcription (STAT)-1 for the induction of iNOS gene expression in response to IFN-gamma has been well defined. Here, we investigated the molecular events responsible for the inhibition of iNOS gene expression by IL-4 in the murine macrophage cell line RAW264.7. Unidirectional deletion analysis on iNOS promoter demonstrated that an IFN-stimulated responsive element (ISRE), contained in the -980 to -765 bp region of the iNOS promoter, may be involved in the IL-4-mediated inhibition of IFN-gamma-inducible iNOS transcription. Accordingly, the IFN-gamma-induced binding activity of IRF-1 to the ISRE sequence was reduced in cells pre-treated with IL-4, while the binding activity of STAT-1 to the STAT-binding element (SBE) within the same region of the iNOS promoter remained unaffected. Moreover, IL-4 even down-regulated IFN-gamma-inducible expression of IRF-1 mRNA. This could be related to a transcriptional mechanism by which IL-4 and IFN-gamma differentially influence the trans-acting activity of the STAT factors binding to SBE within the IRF-1 promoter. SBE is targeted by IFN-gamma-inducible STAT-1 and by IL-4-inducible STAT-6. Although STAT-6 has no trans-acting function on iNOS gene expression, it is able to inhibit the IFN-gamma-induced expression of IRF-1. Thus, IL-4 may down-regulate IFN-gamma-inducible iNOS transcription by activation of STAT-6 which in turn inhibits IRF-1 expression.