Cyclooxygenases (Cox) are rate-limiting enzymes that initiate the conversion of arachidonic acid to prostanoids. Cox-2 is the inducible isoform that is upregulated by proinflammatory agents, initiating many prostanoid-mediated pathological aspects of inflammation. In this study, we demonstrate that interferon (IFN)-gamma alone or in synergy with lipopolysaccharide (LPS) or interleukin 1alpha induces Cox-2 expression in mouse peritoneal macrophages, which is paralleled by changes in Cox-2 protein levels and prostaglandin E(2) (PGE(2)) release. Induction of Cox-2 was abrogated in macrophages that lack IFN regulatory factor (IRF)-1, consistent with an attenuated hepatic mRNA response in IRF-1(-/-) mice injected with LPS. Conversely, the absence of IRF-2 in macrophages resulted in a significant increase in both basal and inducible Cox-2 gene and protein expression as well as IFN-gamma-stimulated PGE(2) release, identifying IRF-2 as negative regulator of this promoter. Two IFN stimulation response elements were identified in the mouse Cox-2 promoter that were highly conserved in the human Cox-2 gene. Both bind endogenous IRF-1 and IRF-2 and regulate transcription in an IRF-1/2-dependent manner. Our data demonstrate conclusively the importance of IFN-gamma as a direct activator and coactivator of the Cox-2 gene, and the central role of IRF-1/2 family members in this process.