Activation of murine peritoneal macrophages or the macrophage cell line RAW264 with IFN‐γ and bacterial lipopolysaccharide promotes a transient up‐regulation of c‐fos family gene expression following inducible NO synthase (iNOS) production. Since introduction of a double mutation into the two AP‐1‐binding sites in the iNOS promoter region reduced the promoter activity to 25% of the authentic one in activated RAW264 cells, the induced c‐Fos/AP‐1 may promote iNOS expression in activated macrophages. Surprisingly, overexpression of c‐fos in activated macrophages completely suppressed the production of iNOS, but not that of IL‐6 and IL‐1β. The regulatory effect was also observed by overexpression of c‐fos, c‐jun or fosB on the promoter activity as deduced from transfection experiments. However, the mutation of AP‐1‐binding sites in the promoter region did not abrogate the regulatory effect of c‐fos and the effect of c‐fos was diminished by co‐transfection with c‐jun, but not with fosB, suggesting no relation between the regulatory effect and a c‐Fos/AP‐1 complex. Expression of NF‐IL6 (C/EBPβ), whose gene product can make a non‐functional heterodimer with c‐Fos family proteins, was transiently induced in activated macrophages. Overexpression of NF‐IL6 in activated RAW264 cells augmented iNOS promoter activity and reduced the regulatory effect of c‐fos overexpression. Thus, overproduction of c‐Fos family proteins acts as a dominant‐negative‐type regulator on iNOS expression in activated macrophages.