A rat aortic banding model of cardiac hypertrophy was used to test the hypothesis that reversible inhibition of mitochondrial respiration by nitric oxide (NO·) elicits a bioenergetic defect in the hypertrophied heart. In support of this hypothesis, the respiration of myocytes isolated from hypertrophied hearts was more sensitive to exogenous NO· (IC 50 200 ± 10 nM vs. 290 ± 30 nM in controls, P = 0.0064). Hypertrophied myocytes also exhibited significantly elevated inducible NO· synthase (iNOS). Consistent with this endogenous source for NO·, the respiration of hypertrophied myocytes was significantly inhibited at physiological O 2 tensions versus controls. Both the nonspecific NOS inhibitor nitro-L-arginine and the iNOS-specific inhibitor N-[3-(aminomethyl)-benzyl]acetamidine·2HCl reversed this inhibition, with no effect on respiration of control myocytes. Consistent with an NO·-mediated mitochondrial dysfunction, the ability of intact perfused hearts to respond to a pacing workload was impaired in hypertrophy, and this effect was reversed by NOS inhibition. We conclude that endogenously generated NO· can modulate mitochondrial function in the hypertrophied heart and suggest that this bioenergetic defect may underlie certain pathological features of hypertrophy. Copyright © 2001 the American Physiological Society.