Reactive oxygen species (ROS) are hypothesized to play a key role in myocardial ischemia-reperfusion (IR) injury after cardiopulmonary bypass in children. Clinical studies in adults and several animal models suggest that myocardial IR injury involves cardiomyocyte apoptosis and necrosis. This study investigated a potential relationship between IR-induced ROS production and neonatal cardiomyocyte apoptosis using both in vitro and ex vivo techniques. For in vitro experiments, embryonic rat cardiomyocytes (H9c2 cells) exposed to hypoxia-reoxygenation (HR) showed a time-dependent increase in gp91 phox (a marker for ROS production by NADPH oxidases), caspase-3 (a key mediator of apoptosis) expression, and a decrease in the glutathione redox ratio. N-acetylcysteine (NAC; 0.25-2 mM), a potent antioxidant, decreased gp91 phox and caspase-3 expression, inhibited apoptosis and restored the glutathione redox ratio. For ex vivo study, IR injury significantly reduced left ventricular (LV) function and increased the expression of gp91 phox and caspase-3 in Langendorff-perfused neonatal (7-14 d) rabbit hearts. NAC (0.4 mM) treatment completely attenuated LV dysfunction after IR. In summary, neonatal myocardial IR injury is associated with an increase in cardiomyocyte oxidative stress and apoptosis. NAC attenuates apoptosis in an in vitro embryonic rat cardiomyocyte model of HR, and myocardial dysfunction in an ex vivo neonatal rabbit model of myocardial IR injury.