BACKROUND: Reactive oxygen species (ROS) mediate anesthetic-induced protection of the heart from ischemia and reperfusion injury (anesthetic preconditioning, APC), but the precise role and mechanism of ROS generation remain unknown. In this study, we tested if a mitochondria-targeted mimetic of superoxide dismutase (mito-tempol, MT) can abolish the reduction in myocardial infarct size afforded by the volatile anesthetic isoflurane. Further, we investigated the mechanism by which isoflurane generates ROS in isolated mitochondria and submitochondrial particles. METHODS: Rats received 0.9% saline (control) or 3.0 mg/kg MT with or without exposure to 1 minimum alveolar concentration isoflurane for 30 min. Myocardial infarction was performed by left anterior descending artery occlusion for 30 min followed by 2 h reperfusion. Infarct size was measured by patent blue and triphenyltetrazolium chloride staining. Mitochondrial ROS production was measured spectrofluorometrically in isolated mitochondria and submitochondrial particles using the fluorescent probe amplex red. The effect of isoflurane on mitochondrial respiratory complex enzyme activities was determined spectrophotometrically in cholic acid-solubilized mitochondria. RESULTS: APC reduced infarct size of the left ventricular area at risk (mean±SD=40±9%) relative to the control (60±4%). MT abolished cardioprotection (60±9%) afforded by APC. Isoflurane enhanced mitochondrial ROS production induced by antimycin A or oxidized ubiquinone in the presence of substrates pyruvate and malate, but not succinate. Isoflurane also produced ROS at Complex I in the absence of any inhibitors in submitochondrial particles. Mitochondrial respiration and electron transport chain complex assays revealed that isoflurane only inhibits complex I activity. CONCLUSIONS: These results highlight that ROS are critical for APC. Moreover, the results indicate that isoflurane produces ROS at complex I and enhances ROS generation at complex III of the respiratory chain via its effect to attenuate complex I activity.