Reactive oxygen species have been known to play a major role in a wide variety of pathophysiologic processes. A new compound, H-2545, based on a 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide structure, has been reported to exhibit antiarrhythmic function as well as favorable antioxidant properties. Studies were performed in an isolated rat heart model to measure the efficacy of H-2545 and its metabolite, H-2954, in preventing ischemia-reperfusion and hydrogen peroxide-induced oxidative myocardial damage: lipid peroxidation, protein oxidation, activity of respiratory complexes, NAD, and high-energy phosphate metabolism. The cardioprotective effects of examined compounds were compared with that of a well-known water-soluble vitamin E analog, Trolox. To determine whether the antioxidant property of H-2545 is due to the pyrroline ring, the scavenger effects of mexiletine and HO-2434 (mexiletine substituted with a pyrroline group) were compared. The results showed that H-2545 decreased significantly the ischemia-reperfusion-induced thiobarbituric acid reactive substance (TBARS) formation, the protein oxidation and ssDNA break formation in perfused rat hearts. H-2545 decreased the NAD loss in postischemic hearts. The activity of respiratory complexes, myocardial energy metabolism, and functional myocardial recovery were also improved during reperfusion by adding H-2545 to the perfusion medium. H-2954 exerted significantly lower protection against ischemia-reperfusion-induced myocardial injury than H-2545, and it was comparable to that of Trolox. Both H-2545 and H-2954 are highly effective against H O -induced oxidative myocardial cell damage. The findings show that substitution of mexiletine with a 2,2,5,5-tetramethyl-pyrroline group (HO-2434) increased its antioxidant and cardioprotective effects. In conclusion, these results suggest that sterically hindered pyrroline derivatives accumulating in membranes can be highly effective at preventing oxidative myocardial cell damage.