Reactive oxygen species have been established as key mediators of cardiac injury following ischemia/reperfusion (I/R). We hypothesized that superoxide formation at different subcellular locations following cardiac I/R injury may differentially regulate cellular responses that determine pathophysiologic outcomes. Recombinant adenoviruses expressing Cu/ZnSOD or MnSOD were utilized to modulate superoxide levels in the cytoplasmic or mitochondrial compartments, respectively, prior to coronary artery I/R injury in the rat heart. Ectopic expression of both MnSOD and Cu/ZnSOD afforded protection from I/R injury, as evidenced by a significant reduction in serum creatine kinase levels, infarct size, malondialdehyde levels, and apoptotic cell death in comparison to controls. MnSOD and Cu/ZnSOD expression also significantly altered the kinetics of NF kappa B and AP-1 activation following I/R injury, characterized by a delayed induction of NF kappa B and abrogated AP-1 response. Western blot analysis of Bcl-2, Bcl-xL, Bad, Caspase 3, PDK1, and phospho-Akt also revealed SOD-mediated changes in gene expression consistent with protection and decreased apoptosis. These findings support the notion that both mitochondrial and cytoplasmic-derived SOD induce changes in AP-1 and NF kappa B activity, creating an antiapoptotic microenvironment within cardiomyocytes that affords protection following I/R injury.