Background - The tolerance of hibernating mammals to cold hypoxia is related to a factor similar to agonists of δ-opioid receptors. This study was designed to assess whether activation of these receptors could reproduce the protection conferred by ischemic preconditioning and whether such cardioprotection was similarly mediated by an opening of ATP-sensitive potassium (K(ATP)) channels. Methods and Results - Thirty-two isolated rat hearts were arrested with and stored in Celsior at 4°C for 5 hours before being reperfused for 2 hours. They were divided into 4 equal groups. Group I hearts served as controls. In group 2, ischemic preconditioning was elicited by two 5-minute global ischemia periods interspersed with 5 minutes of reperfusion before arrest. In group 3, hearts were pharmacologically preconditioned with a 15-minute infusion of the δ-opioid receptor agonist D- Ala2-D-Leu5-enkephalin (DADLE; 200 μmol/L). In group 4, the protocol was similar to group 3 except that infusion of DADLE was preceded by infusion of the K(ATP) blocker glibenclamide (50 μmol/L). The salutary effects of both forms of preconditioning were primarily manifest as a better preservation of diastolic function, a reduced myocardial edema, and reduced creatine kinase leakage. This protection was abolished by administration of glibenclamide before DADLE. Conclusions - These data suggest that activation of δ-opioid receptors improves recovery of cold-stored hearts to a similar extent as ischemic preconditioning, most likely through an opening of K(ATP) channels. This provides a rationale for improving the preservation of hearts for transplantation by pharmacologically duplicating the common pathway to natural hibernation and preconditioning.