The capacity of mitochondria to respond to changes in oxygen delivery has the potential to affect the ability of the heart to tolerate decreased oxygen delivery. Respiration by mitochondria is typically regarded as independent of oxygen tension (pO2) until critically low oxygen concentrations limit the activity of cytochrome oxidase. Paradoxically, there is evidence that cellular and mitochondrial oxygen consumption (respiration) can decline at oxygen tensions well above this critical pO2. We tested the hypothesis that oxygen sensitive decreases in mitochondrial hydrogen peroxide production can decrease cardiac mitochondrial respiration rate. Consistent with previous work, an acute decline in pO2 from 146 mm Hg to 10-13 mm Hg in less than 10 min did not affect mitochondrial respiration rate. In contrast, sustained incubation of mitochondria at a pO2 of 10-13 mm Hg for 30 min caused a 50% decrease in mitochondrial respiration rate. This decrease in mitochondrial respiration rate was mimicked by incubation with the hydrogen peroxide scavenger catalase and the decrease in mitochondrial respiration rate was fully reversible by reintroducing oxygen or by adding hydrogen peroxide. Incubation at low pO2 was also associated with a decreased rate of mitochondrial reactive oxygen species production. These findings indicate that oxygen-dependent decreases in the rate of mitochondrial hydrogen peroxide production can decrease cardiac mitochondrial respiration.