Protection by ischemic preconditioning is lost in cardiomyocytes and hearts of heterozygous connexin 43 deficient (Cx43^+/−) mice. Because connexin 43 (Cx43) is localized in cardiomyocyte mitochondria and mitochondrial Cx43 content is increased with ischemic preconditioning, we now tried to identify a functional defect at the level of the mitochondria in Cx43^+/− mice by use of diazoxide and menadione. Diazoxide stimulates the mitochondrial formation of reactive oxygen species (ROS) and menadione generates superoxide at multiple intracellular sites; both substances elicit cardioprotection through increased ROS formation. ROS formation in response to the potassium ionophore valinomycin was also measured for comparison. Menadione (2 μmol/L) and valinomycin (10 nmol/L) induced similar ROS formation in wild-type (WT) and Cx43^+/− cardiomyocytes. In contrast, diazoxide (200 μmol/L) increased ROS formation by 43±10% versus vehicle in WT, but only by 18±4% in Cx43^+/− cardiomyoctes (P<0.05). Two hour–simulated ischemia and oxygenated, hypo-osmolar reperfusion reduced viability as compared with normoxia (WT: 7±1% versus 39±2%, Cx43^+/−: 8±1% versus 40±3%, P<0.01). Although menadione protected WT and Cx43^+/− cardiomyocytes, diazoxide increased viability (17±2%, P<0.01) in WT, but not in Cx43^+/− (9±1%). Menadione (37 μg/kg i.v.) before 30 minutes coronary occlusion and 2 hour reperfusion reduced infarct size in WT and Cx43^+/− mice (24±4% versus 24±5%). In contrast, diazoxide (5 mg/kg i.v.) reduced infarct size in WT (35±4% versus 55±3% of area at risk, P<0.01), but not in Cx43^+/− mice (56±2% versus 54±3%). Cardiomyocytes of Cx43^+/− mice have a specific functional deficit in ROS formation in response to diazoxide and accordingly less protection.