Rationale: Cyclooxygenase-2 is a key enzyme involved in the inflammatory process that is rapidly induced in macrophages in response to lipopolysaccharide. Carbon monoxide, a byproduct of heme oxygnease-1, can suppress pro-inflammatory response in various in vitro and in vivo models of inflammation. Objective: This study was undertaken to examine whether carbon monoxide can regulate, and if so, delineate the mechanism by which carbon monoxide regulates lipopolysaccharide-induced cyclooxygenase-2 expression in macrophages. Methods: RAW 264.7 murine macrophages were stimulated with lipopolysaccharide (0-10 ng/ml) with or without carbon monoxide (500 ppm). Northern and western blot analysis was done. Progstaglandin E₂ and nitrite concentration was measured from cell culture supernatant. Electrophoretic mobility shift assay was performed to assess nuclear factor binding. Measurement and Main results: Carbon monoxide down-regulated lipopolysaccharide-induced cyclooxygenase-2 mRNA and protein expression. Carbon monoxide also inhibited lipopolysaccharide-induced prostaglandin E₂ secretion (p<0.05). Carbon monoxide also decreased lipopolysaccharide-induced CCAAT/enhancer binding protein and protein expression in lipopolysaccharide-treated RAW 264.7 cells. Gel shift analysis revealed that carbon monoxide treatment decreased lipopolysaccharide-induced activation of protein binding to CCAAT/enhancer binding protein consensus oligonucleotides of murine cycloosygenase-2 promoter. Carbon monoxide also decreased lipopolysaccharide-induced nitric oxide synthase-2 protein expression and nitrite production and decreased lipopolysaccharide-induced activation of protein binding to CCAAT/enhancer binding protein consensus oligonucleotides of murine nitric oxide synthase-2 promotor. Conclusion: Carbon monoxide may act as an important regulator of inflammation by virtue of its ability to regulate CCAAT/enhancer binding proteins.