Rationale: Sepsis produces significant mitochondrial and contractile dysfunction in the heart but the role of superoxide derived free radicals in the genesis of these abnormalities is not completely understood. Objectives: The study was designed to test the hypothesis that superoxide scavenger administration prevents endotoxin-induced cardiac mitochondrial and contractile dysfunction. Methods: Four groups of rats were studied and animals were injected with either saline, endotoxin, endotoxin plus polyethylene glycol adsorbed superoxide dismutase (PEG-SOD, a free radical scavenger), or PEG-SOD alone. Animals were sacrificed 48 hours after injections. We then measured cardiac mitochondrial generation of reactive oxygen species (ROS), formation of free radical reaction products (protein carbonyls, lipid aldehydes, nitrotyrosine), mitochondrial function and cardiac contractile function. Measurements and Main Results: Endotoxin elicited increases in cardiac mitochondrial ROS formation (p<0.001), increases in cardiac levels of free radical reaction products, reductions in mitochondrial ATP generation (p<0.001), and decrements in cardiac pressure generating capacity (p<0.01). Administration of PEG-SOD blocked formation of free radical reaction products, prevented mitochondrial dysfunction and preserved cardiac contractility. For example, mitochondrial ATP generation was 923±50, 392±32, 753±25, and 763±36 nmol/min/mg, respectively, for control, endotoxin, endotoxin + PEG-SOD and PEG-SOD groups (p<0.001). In addition, cardiac systolic pressure generation at a diastolic pressure of 15 mm Hg averaged 110±11, 66±7, 129±10 and 124±5 mm Hg, respectively for control, endotoxin, endotoxin + PEG-SOD and PEG-SOD groups (p<0.01). Conclusion: These data indicate that superoxide derived oxidants play a critical role in the development of cardiac mitochondrial and contractile dysfunction in endotoxin-induced sepsis.