Tissue changes consistent with oxidative damage in hypoxic/reoxygenated heart tissue have not been well documented. We recently reported that oxidative perturbations were evident in isolated-perfused rat heart tissue subjected to as little as 10 min hypoxia and that these changes were not exacerbated by reoxygenation. The mechanism and species specificity of this finding is not known. Rabbit hearts, which lack measurable xanthine oxidase activity, were examined for evidence of hypoxia-induced injury. The release of lactate dehydrogenase into the coronary effluent gradually increased during the retrograde perfusion of isolated rabbit hearts with hypoxic medium (containing 10 mM glucose and 2.5 mM calcium), and was slightly enhanced upon reoxygenation after 60 min hypoxia. Cardiac glutathione content decreased significantly while glutathione disulfide, protein-glutathione mixed disulfides, thiobaribturic acid reactive substances (TBARS), and protein carbonyl contents increased significantly after 60 min of hypoxia, compared to oxygenated controls. These values were unaltered after 4 min of reoxygenation except for a loss of TBARS. The oxidative changes observed in hypoxic rabbit hearts may be caused by energy deficiency impairing normal reductive processes or by the generation of reactive oxygen species as a result of abnormal cell functions, but cannot be related to xanthine oxidase activity.