Necrostatin (Nec-1) protects against ischemia-reperfusion (IR) injury in both brain and heart. We have previously reported in this journal that necrostatin can delay opening of the mitochondrial permeability transition pore (MPTP) in isolated cardiomyocytes. The aim of the present study was to investigate in more detail the role played by the MPTP in necrostatin-mediated cardioprotection employing mice lacking a key component of the MPTP, namely cyclophilin-D. Anaesthetized wild type (WT) and cyclophilin-D knockout (Cyp-D-/-) mice underwent an open-chest procedure involving 30 minutes of myocardial ischemia and 2 hours of reperfusion, with subsequent infarct size assessed by triphenyltetrazolium staining. Nec-1, given at reperfusion, significantly limited infarct size in WT mice (17.7±3% vs. 54.3±3%, P0.05). In conclusion, the data obtained in Cyp-D−/− mice provide further evidence that Nec-1 protects against myocardial IR injury by modulating MPTP opening at reperfusion.