Cardiac hypertrophy is a significant risk factor for the development of congestive heart failure (CHF). Mitochondrial defects are reported in CHF, but no consistent mitochondrial alterations have yet been identified in hypertrophy. In this study selective metabolic inhibitors were used to determine thresholds for respiratory inhibition and to reveal novel mitochondrial defects in hypertrophy. Cardiac hypertrophy was produced in rats by aortic banding. Mitochondria were isolated from left ventricular tissue and the effects of inhibiting respiratory complexes I and IV on mitochondrial oxygen consumption were measured. At 8 weeks post-surgery, 65 ± 2% complex IV inhibition was required to inhibit respiration half maximally in control mitochondria. In contrast, only 52 ± 6% complex IV inhibition was required to inhibit respiration half maximally in mitochondria from hypertrophied hearts (P = 0.046). This effect persisted at 22 weeks post-surgery and was accompanied by a significant upregulation of inducible nitric oxide synthase (iNOS, 3.0 ± 0.7-fold, P = 0.006). We conclude that respiration is more sensitive to complex IV inhibition in hypertrophy. Nitric oxide is a well documented inhibitor of complex IV, and thus the combination of increased NO from iNOS and an increased sensitivity to inhibition of one of its targets could result in a bioenergetic defect in hypertrophy that may be a harbinger of CHF. © 2000 Academic Press.