The aim of the study was to examine the cardioprotective effect of morphine and Delta 2 opioid d-Ala2-Leu5 enkephalin (DADLE) administered, at early reoxygenation, in isolated human myocardium exposed to hypoxia-reoxygenation. Then, we tested the involvement of mitochondrial permeability transition pore in morphine and DADLE-induced postconditioning. Human right atrial trabeculae were obtained during cardiac surgery (coronary artery bypass and aortic valve replacement). Isometrically contracting isolated human right atrial trabeculae were exposed to 30-min hypoxia and 60-min reoxygenation (control group). In treatment groups, morphine 0.5 μmol, DADLE 10 nmol, DADLE 50 nmol and DADLE 100 nmol were administered during the first 15 min of reoxygenation. In two additional groups, morphine and DADLE 100 nmol were administered in the presence of atractyloside 50 μmol, the mitochondrial permeability transition pore opener. The force of contraction at the end of 60-min reoxygenation period (FoC(60) expressed as % of baseline) was compared (mean ± standard deviation) between the groups by an analysis of variance. Morphine (FoC(60): 81 ± 9% of baseline), DADLE 50 nmol (FoC(60): 76 ± 11% of baseline) and DADLE 100 nmol (FoC(60): 81 ± 4% of baseline) increased significantly (P < 0.001) the FoC(60) as compared with the control group (FoC(60): 53 ± 3% of baseline). DADLE 10 nmol did not modify the FoC(60) (50 ± 9% of baseline; P = 0.60 versus control group). The enhanced recovery of FoC(60) induced by morphine and DADLE 100 nmol were abolished in the presence of atractyloside (FoC(60): respectively 57 ± 6% and 44 ± 7% of baseline; P < 0.001). In conclusion, the administration of morphine and DADLE, in early reoxygenation period, protected human myocardium, in vitro, against hypoxia-reoxygenation injury, at least in part, by the inhibition of mitochondrial permeability transition pore opening.