—Experimental and clinical studies have suggested an increased production of reactive oxygen species (ROS) in the failing myocardium. The present study aimed to obtain direct evidence for increased ROS and to determine the contribution of superoxide anion (·O₂⁻), H₂O₂, and hydroxy radical (·OH) in failing myocardial tissue. Heart failure was produced in adult mongrel dogs by rapid ventricular pacing at 240 bpm for 4 weeks. To assess the production of ROS directly, freeze-clamped myocardial tissue homogenates were reacted with the nitroxide radical, 4-hydroxy-2,2,6,6,-tetramethyl-piperidine-N-oxyl, and its spin signals were detected by electron spin resonance spectroscopy. The rate of electron spin resonance signal decay, proportional to ·OH level, was significantly increased in heart failure, which was inhibited by the addition of dimethylthiourea (·OH scavenger) into the reaction mixture. Increased ·OH in the failing heart was abolished to the same extent in the presence of desferrioxamine (iron chelator), catalase (H₂O₂ scavenger), and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron; LaMotte) (·O₂⁻ scavenger), indicating that ·OH originated from H₂O₂ and ·O₂⁻. Further, ·O₂⁻ produced in normal myocardium in the presence of antimycin A (mitochondrial complex III inhibitor) could reproduce the increase of H₂O₂ and ·OH seen in the failing tissue. There was a significant positive relation between myocardial ROS level and left ventricular contractile dysfunction. In conclusion, in the failing myocardium, ·OH was produced as a reactive product of ·O₂⁻ and H₂O₂, which might play an important role in left ventricular failure.