Objective: Previously we demonstrated that a novel opiate peptide, 2���,6���-dimethyltyrosine(Dmt)-D-Arg-Phe-Lys-NH2, provided cardioprotection against myocardial stunning in vivo. We subsequently showed that this peptide targeted mitochondria and can scavenge reactive oxygen species (ROS). The objective of this study was to determine the role of opioid versus antioxidant activity in cardioprotection. Methods: We compared two mitochondria-targeted peptide analogues that lacked opioid activity: SS-31 (D-Arg-Dmt-Lys-Phe-NH2) and SS-20 (Phe-D-Arg-Phe-Lys- NH2). They differ in that only SS-31 has scavenging ability. Rats (n = 8/group) were randomized to SS-31, SS-20, or placebo. The drugs (3 mg/kg) or saline was administered intraperitoneally (IP) 30 minutes prior to ligation of left anterior descending artery for 60 minutes, and another dose given IP 5 minutes before reperfusion for 60 minutes. Study endpoints included myocardial infarct size, cardiac arrhythmia, and myocardial lipid peroxidation. Results: The area at risk was similar among the groups. However, the infarct area/area at risk was significantly smaller in the treatment groups (53.91.1% in SS-31 group, 47.1 짹 1.4% in SS-20 group, versus 59.9짹1% in the controls, p<0.01). Lipid peroxidation was significantly reduced by both SS-31 and SS-20 treatment. Arrhythmia occurred only during early period of coronary occlusion and was less frequent and less severe in the peptide treatment groups versus the controls (Lambeth score 5 points, 3 points, versus 13 points in the contols, p<0.05). Conclusions: This study shows that pretreatment with both SS-31 and SS-20 significantly reduced myocardial lipid peroxidation and infarct size in ischemia reperfusion injury, and suggests that the cardioprotective properties of (Dmt)-D-Arg-Phe-Lys-NH2) was primarily mediated by its antioxidant properties. Since SS-20 does not scavenge ROS, it most likely reduces ROS production during ischemia-reperfusion.