Manganese-containing superoxide dismutase (Mn-SOD) plays a critical role in guarding against mitochondrial oxidative stress. Abnormal myocardial mitochondrial metabolism of reactive oxygen species plays an important role in the pathogenesis of diabetic cardiac dysfunction. We hypothesised that chronic treatment with N-acetylcysteine, an antioxidant and glutathione (GSH) precursor, would normalize hyperglycemia induced inactivation of Mn-SOD and attenuate myocardial dysfunction. Control and streptozotozin-induced diabetic rats were treated or untreated with N-acetylcysteine in drinking water for 8 weeks, initiated 1 week after streptozotozin injection. Myocardial performance was determined using the isolated perfused working heart preparation. Myocardial Mn-SOD activity, but not Mn-SOD protein expression, in diabetic rats was significantly reduced while levels of oxidative stress as determined by myocardial free 15-F2t-isoprostane were increased in diabetic rats and were normalized by N-acetylcysteine treatment. However, compensatory increases in myocardial Cu/Zn-SOD and GSH content were seen in diabetic rats accompanied by an increase in tissue antioxidant capacity as compared to control. N-acetylcysteine abolished the compensatory increase in myocardial Cu/Zn-SOD. The left ventricular developed pressure and rates of left ventricular pressure development and relaxation were decreased in diabetic rats as compared to control. These effects were attenuated, but not prevented by N-acetylcysteine treatment. N-acetylcysteine attenuation of diabetic myocardial dysfunction could be attributed to the restoration of myocardial Mn-SOD activity.