Cardiac ischemia decreases complex III activity, cytochrome c content, and respiration through cytochrome oxidase in subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). The reversible blockade of electron transport with amobarbital during ischemia protects mitochondrial respiration and decreases myocardial injury during reperfusion. These findings support that mitochondrial damage occurs during ischemia and contributes to myocardial injury during reperfusion. The current study addressed if ischemic damage to the electron transport chain (ETC) increased the net production of reactive oxygen species (ROS) from mitochondria. SSM and IFM were isolated from 6 mo. Fisher 344 rat hearts following 25 min. global ischemia or following 40 min. of perfusion alone as controls. H₂O₂ release from SSM and IFM was measured using the amplex red assay. With glutamate as a complex I substrate, the net production of H₂O₂ was increased by 178±14% and 179±17% in SSM and IFM (n=9) following ischemia compared to controls (n=8). With succinate as substrate in the presence of rotenone, H₂O₂ increased by 272±22% and 171±21% in SSM and IFM after ischemia. Inhibitors of electron transport were used to assess maximal ROS production. Inhibition of complex I with rotenone increased H₂O₂ tion by 179±24% and 155±14% in SSM and IFM following ischemia. Ischemia also increased the antimycin A-stimulated production of H₂O₂ from complex III. Thus, ischemic damage to the ETC increased both the capacity and the net production of H₂O₂ from complex I and complex III and sets the stage for cardiac injury during reperfusion due to an increase in ROS production.