Aim: To examine if isoflurane preconditioning can attenuate ischemia/reperfusion injury by reducing cytochrome c release from inner mitochondrial membrane.Methods: Isolated hearts of Sprague-Dawley rats were perfused on Langendorff apparatus.Hearts were randomly assigned to a non-treated group (CON group,n=12) or three isoflurane preconditioning groups (0.5% ISC group, 1.0% ISC group,and 2.0% ISC group; n=12).In the latter three groups, isoflurane was given at concentrations of 0.5%, 1.0%, and 2.0% for 15 min with 15-min washout before 30-min ischemia.Subsarcolemmal mitochondria of the myocardium were isolated after 60-min reperfusion.Hemodynamics of the each heart was recorded, infarct size of the hearts and contents of cytosolic cytochrome or mitochondrial cytochrome c were measured at the end of reperfusion.Morphology of isolated mitochondria in the four groups was evaluated, respectively.Results: Compared with the CON group, cytosolic cytochrome c in 0.5% ISC group, 1.0% ISC group, and 2.0% ISC group were significantly decreased along with a significant increase of mitochondrial cytochrome c.Infarct size of the hearts in the four groups were 56%±12%, 41%±12%, 32%±7% and 33%±11%, respectively.The values of the three isoflurane preconditioning groups were significantly lower than that of the CON group (P＜0.05).Isoflurane exposure before ischemia can attenuate the change of morphology of mitochondria after reperfusion.The effects of 2.0% isoflurane on reducing cytochrome c release were more remarkable than 0.5% and 1.0%concentrations of isoflurane.Conclusion: Myocardioprotective effects of isoflurane preconditioning were associated with attenuation of cytochrome c loss from the inner membrane of subsarcolemmal mitochondria.