IFN-gamma induces cyclooxygenase (COX)-2 expression and PG production in mouse macrophage cells. IFN-gamma activates COX-2 promoter-driven transcription. Deletion of the IFN sequence regulatory element (ISRE) I -1541/-1522 and ISRE II -1215/-1206 sites of the mouse COX-2 promoter minimally decrease this IFN-gamma induction. In contrast, deletion of the -965/-150 region from the COX-2 promoter abrogated IFN-gamma induction. In this region a NF-kappaB site has been described and mutation of this site impairs the induction of the full COX-2 promoter by IFN-gamma. Moreover, IFN-gamma induction of the COX-2 promoter was also strongly reduced by transfection of plasmid encoding the NF-kappaB inhibitor, IkappaBalpha. Interestingly, IFN-gamma induction of the COX-2 and PGE(2) synthesis was absent in macrophages from TNF(-/-) mice, and neutralizing anti-TNF Abs inhibited COX-2 promoter induction by IFN-gamma in RAW 264.7 macrophages. Moreover, NF-kappaB activity was induced late after stimulation with IFN-gamma correlating with the effect of autocrine TNF, and this NF-kappaB activation was absent in macrophages from TNF(-/-) mice. Taken together our results suggest a model in which IFN-gamma-induced TNF activates NF-kappaB, which is required for full COX-2 expression.