Calcium-independent phospholipase A 2 ␥ (iPLA 2 ␥) is a mitochondrial enzyme that produces lipid second messengers that facilitate opening of the mitochondrial permeability transition pore (mPTP) and contribute to the production of oxidized fatty acids in myocardium. To specifically identify the roles of iPLA 2 ␥ in cardiac myocytes, we generated cardiac myocyte-specific iPLA 2 ␥ knock-out (CMiPLA 2 ␥KO) mice by removing the exon encoding the active site serine (Ser-477). Hearts of CMiPLA 2 ␥KO mice exhibited normal hemodynamic function, glycerophospholipid molecular species composition, and normal rates of mitochondrial respiration and ATP production. In contrast, CMiPLA 2 ␥KO mice demonstrated attenuated Ca 2؉ -induced mPTP opening that could be rapidly restored by the addition of palmitate and substantially reduced production of oxidized polyunsaturated fatty acids (PUFAs). Furthermore, myocardial ischemia/reperfusion (I/R) in CMiPLA 2 ␥KO mice (30 min of ischemia followed by 30 min of reperfusion in vivo) dramatically decreased oxidized fatty acid production in the ischemic border zones. Moreover, CMiPLA 2 ␥KO mice subjected to 30 min of ischemia followed by 24 h of reperfusion in vivo developed substantially less cardiac necrosis in the area-atrisk in comparison with their WT littermates. Furthermore, we found that membrane depolarization in murine heart mitochondria was sensitized to Ca 2؉ by the presence of oxidized PUFAs. Because mitochondrial membrane depolarization and calcium are known to activate iPLA 2 ␥, these results are consistent with salvage of myocardium after I/R by iPLA 2 ␥ loss of function through decreasing mPTP opening, diminishing production of proinflammatory oxidized fatty acids, and attenuating the deleterious effects of abrupt increases in calcium ion on membrane potential during reperfusion.The salvage of jeopardized regions of myocardium during ischemia/reperfusion (I/R) 3 has been a long-standing goal of heart research. Because mortality and morbidity are related to infarct size, a variety of hemodynamic, metabolic, and pharmacological approaches have been used to reduce the severity of myocardial infarction during ischemia (1-3). Recent studies have accumulated evidence that the irreversible opening of the mitochondrial permeability transition pore (mPTP) upon oxidative stress is a principal mechanism of apoptotic/necrotic cardiac cell death accounting for the majority of I/R injury (4 -6). Although therapies for acute ischemia (e.g. reperfusion) have been extensively studied, at present there is no therapy for attenuating mPTP opening during reperfusion of ischemic zones in myocardium.Although the precise chemical composition of the mPTP is incompletely understood (6), a variety of initiators and modulators of mPTP opening has been identified (7,8). For example, during reperfusion, the reoxygenation of ischemic tissue results in mitochondrial Ca 2ϩ overload and renormalization of intracellular and matrix pH, which are accompanied by the prodigious generation of reactive oxygen s...