Reperfusion of ischemic tissue results in the generation of reactive oxygen species that contribute to tissue injury. The sources of reactive oxygen species in reperfused tissue are not fully characterized. We hypothesized that the small GTPase Rac1 mediates the oxidative burst in reperfused tissue and thereby contributes to reperfusion injury. In an in vivo model of mouse hepatic isch‐emia/reperfusion injury, recombinant adenoviral expression of a dominant negative Rac1 (Rac1N17) completely suppressed the ischemia/reperfusion‐induced production of reactive oxygen species and lipid peroxides, activation of nuclear factor‐kappa B, and resulted in a significant reduction of acute liver necrosis. Expression of Rac1N17 also suppressed ischemia/reperfusion‐induced acute apoptosis. The protection offered by Rac1N17 was also evident in knockout mice deficient for the gp91phox component of the phagocyte NADPH oxidase. This work demonstrates the crucial role of a Rac1‐regulated oxidase in mediating the production of injurious reactive oxygen species, which contribute to acute necrotic and apoptotic cell death induced by isch‐emia/reperfusion in vivo. Targeted inhibition of this oxidase, which is distinct from the phagocyte NADPH oxidase, should provide a new avenue for in vivo therapy aimed at protecting organs at risk from ischemia/reperfusion injury.—Ozaki, M., Deshpande, S. S., Angkeow, P., Bellan, J., Lowenstein, C. J., Dinauer, M. C., Goldschmidt‐Clermont, P. J., Irani, K. Inhibition of the Rac1 GTPase protects against nonlethal ischemia/reperfusion‐induced necrosis and apoptosis in vivo. FASEB J. 14, 418—429 (2000)