Recently, we reported that exogenous administration of Met5-enkephalin (ME) for 24 h reduces infarct size after ischemia-reperfusion in rabbits. In the present study, we tested whether ME-induced cardioprotection is exhibited in murine hearts and whether chronic infusion of this peptide can render hearts tolerant to ischemia. Barbiturate-anesthetized open-chest mice (C57BL/6J) were subjected to regional myocardial ischemia-reperfusion (45 min of occlusion and 20 min of reperfusion). Mice received saline vehicle or ME for 24 h or 2 wk before undergoing regional myocardial ischemia-reperfusion or for 24 h followed by a 24-h delay before regional myocardial ischemia-reperfusion. Infarct size was measured with propidium iodide and is expressed as a percentage of the area at risk. Infarcts were smaller after infusion of ME for 24 h than with vehicle control: 49.2 ± 9.0% vs. 22.2 ± 3.2% ( P < 0.01). In contrast, administration of ME for 2 wk failed to elicit cardioprotection: 36.5 ± 9.1% and 41.4 ± 8.2% for control and ME, respectively ( P = not significant). When a 24-h delay was imposed between the end of drug treatment and the onset of the ischemic insult, cardioprotection was lost: 38.5 ± 6.1% and 42.8 ± 6.6% for control and ME, respectively ( P = not significant). Chronic sustained exogenous infusion of the endogenously produced opioid peptide ME is associated with loss of the cardioprotection that is observed with 24 h of infusion. Furthermore, in this in vivo murine model, ME failed to induce delayed tolerance to myocardial ischemia-reperfusion.