Aim:Attenuation of mitochondrial Ca~(2+)([Ca~(2+)]_m),but not cytosolic Ca~(2+)([Ca~(2+)]_c),overload improves contractile recovery.We hypothesized that attenuation of[Ca~(2+)]_m,but not[Ca~(2+)]_c,overload confers cardioprotection against ischemia/reperfusion-induced injury.Methods:Infarct size from isolated perfused rat heart,cell viability,and electrically-induced Ca~(2+)transient in isolated rat ventricularmyocytes were measured.We determined the effects of BAPTA-AM,a Ca~(2+)chelator,at concentrations that abolish the overload of both[Ca~(2+)]_c and[Ca~(2+)]_m,and ruthenium red,an inhibitor of mitochondrial uniporter of Ca~(2+)transport,atconcentrations that abolish the overload of[Ca~(2+)]_m,but not[Ca~(2+)]_c,on cardiacinjury induced by ischemia/reperfusion.Results:Attenuation of both[Ca~(2+)]_mand[Ca~(2+)]_c by BAPTA-AM,and attenuation of[Ca~(2+)]_m,but not[Ca~(2+)]_c,overloadby ruthenium red,reduced the cardiac injury observations,indicating the impor-tance of[Ca~(2+)]_m in cardioprotection and contractile recovery in response to is-chemia/reperfusion.Conclusion:The study has provided unequivocal evidenceusing a cause-effect approach that attenuation of[Ca~(2+)]_m,but not[Ca~(2+)]_c,over-load is responsible for cardioprotection against ischemia/reperfusion-inducedinjury.We also confirmed the previous observation that attenuation of[Ca~(2+)]_m,but not[Ca~(2+)]_c,by ruthenium red improves contractile recovery following ischemia/reperfusion. Aim: Attenuation of mitochondrial Ca ~ (2 +) ([Ca ~ (2 +)] _ m) but not cytosolic Ca ~ (2 +) ([Ca ~ hypothesized that attenuation of [Ca ~ (2 +)] _ m, but not [Ca ~ (2 +)] _ c, overload confers cardioprotection against ischemia / reperfusion-induced injury. Methods: Infarct size from isolated perfused rat heart, cell viability, and electrically-induced Ca ~ (2 +) transient in isolated rat ventricular myocytes were measured. We determined the effects of BAPTA-AM, a Ca ~ (2+) chelator, at concentrations that abolish the overload of both [Ca ~ )] c and [Ca ~ (2 +)] _ m, and ruthenium red, an inhibitor of mitochondrial uniporter of Ca ~ (2+) transport, atconcentrations that that abolish the overload of [Ca ~ (2 +)] _ m, but not Results: Attenuation of both [Ca ~ (2 +)] _ mand [Ca ~ (2 +)] _ c by BAPTA-AM, and attenuation of [Ca ~ Ca2 +] _ c, overloadby ruthenium red, reduced the cardiac injury observations, indicating the impor tance of [Ca ~ (2 +)] _ m in car dioprotection and contractile recovery in response to is-chemia / reperfusion. Conlusion: The study has provided unequivocal evidence using a cause-effect approach that attenuation of [Ca ~ (2 +)] _ m, but not [Ca ~ (2 +)] _ c , over-load is responsible for cardioprotection against ischemia / reperfusion-induced in jeury. We also confirmed the previous observation that attenuation of [Ca ~ (2 +)] _ m, but not [Ca ~ (2 +)] _ c, by ruthenium red improves contractile recovery following ischemia / reperfusion.