Mitochondria are an important source of reactive oxygen species (ROS), implicated in ischemia/reperfusion injury. When isolated from ischemic myocardium, mitochondria demonstrate increased ROS production as a result of damage to electron transport complexes. To investigate the mechanisms, we studied effects of hypoxia/reoxygenation on ROS production by isolated energized heart mitochondria. ROS production, tracked using Fe(2+)-catalyzed, H(2)O(2)-dependent H(2)DCF oxidation or Amplex Red, was similar during normoxia and hypoxia but markedly increased during reoxygenation, in proportion to the duration of hypoxia. In contrast, if mitochondria were rapidly converted from normoxia to near-anoxia ([O(2)], <1 micromol/L), the increase in H(2)DCF oxidation rate during reoxygenation was markedly blunted. To elicit the robust increase in H(2)DCF oxidation rate during reoxygenation, hypoxia had to be severe enough to cause partial, but not complete, respiratory chain inhibition (as shown by partial dissipation of membrane potential and increased NADH autofluorescence). Consistent with its cardioprotective actions, nitric oxide ( O) abrogated increased H(2)DCF oxidation under these conditions, as well as attenuating ROS-induced increases in matrix [Fe(2+)] and aconitase inhibition caused by antimycin. Collectively, these results suggest that (1) hypoxia that is sufficient to cause partial respiratory inhibition is more damaging to mitochondria than near-anoxia; and (2) O suppresses ROS-induced damage to electron transport complexes, probably by forming O-Fe(2+) complexes in the presence of glutathione, which inhibit hydroxyl radical formation.