Ischemic preconditioning (IPC) before sustained ischemia decreases myocardial infarct size mediated in part via protection of cardiac mitochondria. Reversible blockade of electron transport at complex I immediately before sustained ischemia also preserves mitochondrial respiration and decreases infarct size. We proposed that IPC would attenuate electron transport from complex I as a potential effector mechanism of cardioprotection. Isolated, Langendorff-perfused rat hearts underwent IPC (3 cycles 5 min 37°C global ischemia and 5 min reperfusion) or were perfused for 40 min without ischemia as controls. Subsarcolemmal (SSM) and interfibrillar (IFM) populations of mitochondria were isolated. IPC did not decrease ADP-stimulated respiration measured in intact mitochondria using substrates that donate reducing equivalents to complex I. Maximally-expressed complex I activity measured as rotenone sensitive NADH: ubiquinone oxidoreductase in detergent-solubilized mitochondria was also unaffected by IPC. Thus, the protection of IPC does not occur as a consequence of a partial decrease in complex I activity leading to a decrease in integrated respiration through complex I. IPC and blockade of electron transport both converge upon mitochondria as effectors of cardioprotection, however each modulates mitochondrial metabolism during ischemia by different mechanisms in order to achieve cardioprotection.