Abstract Background The nature of oxidative stress and the activity of antioxidant enzyme systems are incompletely characterized in the failing human heart. Methods and Results We obtained ventricular myocardium from failing, explanted human hearts in patients with nonischemic dilated cardiomyopathy at the time of heart transplant to examine whether reactive oxygen species (ROS) production and antioxidant enzyme activity or expression were altered in end-stage human heart failure. Nonfailing myocardium was obtained from organ donors who were not eligible for transplantation. Electroparamagnetic resonance (EPR) with the O 2 − spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide demonstrated that formation of superoxide anion was increased more than 2-fold in the failing ( P < .001 vs. nonfailing) myocardium. Manganese superoxide dismutase (MnSOD) mRNA and catalase mRNA expression were increased by 52% ( P = .05) and 116% ( P < .05), respectively, in failing vs. nonfailing hearts. Copper-zinc superoxide dismutase (CuZnSOD) mRNA and glutathione peroxidase-1 (GPx-1) mRNA were unchanged. The expression of MnSOD, CuZnSOD, and catalase mRNA showed moderate correlation, suggesting coordinate regulation of gene expression. Activity was no different with regard to catalase, GPx-1, and glucose-6-phosphate dehydrogenase. MnSOD activity accounted for approximately 90% of total SOD activity, and was markedly decreased in failing hearts (by 61%, P < .05). MnSOD protein expression by western blot analysis was decreased in the failing group ( P < .05 vs. nonfailing). Conclusion The decrease in MnSOD activity in failing myocardium, in the setting of increased mRNA expression, may reflect decreased translation or processing, or a posttranslational modification of MnSOD. The increase in MnSOD mRNA in failing hearts is consistent with the thesis that there is increased oxidative stress in failing myocardium that leads to increase transcription of antioxidant enzymes. The source of this direct measure of ROS is likely superoxide. These observations have implications for the pathophysiology and treatment of heart failure.