Increased oxidative stress is a known cause of cardiac dysfunction in diabetic animals and patients, but the sources of reactive oxygen species (e.g., superoxide anion (O₂^-)), and the mechanisms underlying O₂^- production in diabetic hearts are not clearly understood. Our aim was to determine whether NADPH oxidase (Nox) is a source of O₂^- and whether glucose-6-phosphate dehydrogenase (G6PD)-derived NADPH plays a role in augmenting O₂^- generation in diabetes. We assessed cardiac function, Nox and G6PD activities, NADPH levels, and the activities of antioxidant enzymes in heart homogenates from young (9-11 weeks old) Zucker lean and obese (fa/fa) rats. We found that myocardial G6PD activity was significantly higher in fa/fa than lean rats, whereas superoxide dismutase and glutathione peroxidase activities were decreased (P<0.05). O₂^- levels were elevated (70-90%; P<0.05) in the diabetic heart, and this elevation was blocked by the Nox inhibitor gp-91^ds-tat (50 µM) or by the mitochondrial respiratory chain inhibitors antimycin (10 µM) and rotenone (50 µM). Inhibition of G6PD by 6-aminonicotinamide (5 mM) and dihydroepiandrosterone (100 µM) also reduced (P<0.05) O₂^- production. Notably, the activities of Nox and G6PD in the fa/fa rat heart were inhibited by chelerythrine, a protein kinase C inhibitor. Although we detected no changes in stroke volume, cardiac output or ejection fraction, left ventricular diameter was slightly increased during diastole and systole, and left ventricular posterior wall thickness was decreased during systole (P<0.05) in Zucker fa/fa rats. Our findings suggest that in a model of severe hyperlipidema and hyperglycemia Nox-derived O₂^- generation in the myocardium is fueled by elevated levels of G6PD-derived NADPH. Similar mechanisms were found to activate O₂^- production and induce endothelial dysfunction in aorta. Thus, G6PD may be a useful therapeutic target for treating the cardiovascular disease associated with type 2 diabetes.