In the failing heart, NADPH oxidase and uncoupled NO synthase utilize cytosolic NADPH to form superoxide. NADPH is supplied principally by the pentose phosphate pathway, whose rate-limiting enzyme is glucose 6-phosphate dehydrogenase (G6PD). Therefore, we hypothesized that cardiac G6PD activation drives part of the excessive superoxide production implicated in the pathogenesis of heart failure. Pacing-induced heart failure was performed in eight chronically instrumented dogs. Seven normal dogs served as control. End-stage failure occurred after 28 ± 1 days of pacing, when left ventricular end-diastolic pressure reached 25 mm Hg. In left ventricular tissue homogenates, spontaneous superoxide generation measured by lucigenin (5 μM) chemiluminescence was markedly increased in heart failure (1338 ± 419 vs. 419 ± 102 AU/mg protein, P < 0.05), as were NADPH levels (15.4 ± 1.5 vs. 7.5 ± 1.5 μmol/gww, P < 0.05). Superoxide production was further stimulated by the addition of NADPH. The NADPH oxidase inhibitor gp91ds-tat (50 μM) and the NO synthase inhibitor l-NAME (1 mM) both significantly lowered superoxide generation in failing heart homogenates by 80% and 76%, respectively. G6PD was upregulated and its activity higher in heart failure compared to control (0.61 ± 0.10 vs. 0.24 ± 0.03 nmol/min/mg protein, P < 0.05), while superoxide production decreased to normal levels in the presence of the G6PD inhibitor 6-aminonicotinamide. We conclude that the activation of myocardial G6PD is a novel mechanism that enhances NADPH availability and fuels superoxide-generating enzymes in heart failure. © 2006 Elsevier Inc. All rights reserved.