Carbon monoxide (CO), which is produced endogenously in the breakdown of heme, has been recognized as an important physiological second messenger similar to NO. Additionally, pharmacological delivery of CO is protective in numerous models of injury, including ischemia/reperfusion, transplantation, hemorrhagic shock, and endotoxemia. However, the mechanism of action of CO is only partially elucidated focused primarily on how it modulates the cellular response to stress. The purpose of these investigations is to test the hypothesis that CO acts via inhibition of cytochrome c oxidase leading to the generation of low levels of reactive oxygen species (ROS) that in turn mediate subsequent adaptive signaling. We show here that CO increases ROS generation in RAW 264.7 cells, which is inhibited by antimycin A and is absent in respiration‐deficient p° cells. CO inhibits cytochrome c oxidase, while maintaining cellular ATP levels and increasing mitochondrial membrane potential. The addition of antioxidants or inhibition of complex III of the electron transport chain by antimycin A attenuates the inhibitory effects of CO on lipopolysaccharide (LPS)‐induced TNF‐α and blocked CO‐induced p38 MAPK phosphorylation, which we previously have shown to be important in the anti‐inflammatory effects of CO.—Zuckerbraun, B. S., Chin, B. Y., Bilban, M., de Costa d'Avila, J., Rao, J., Billiar, T. R., Otterbein, L. E. Carbon monoxide signals via inhibition of cytochrome c oxidase and generation of mitochondrial reactive oxygen species. FASEB J. 21, 1099–1106 (2007)