We investigated the effect on mitochondrial Ca²⁺ of SEA0400, an inhibitor of the Na⁺/Ca²⁺ exchanger (NCX) which reduces mitochondrial Ca²⁺ overload during myocardial ischemia, in digitonin-permeabilized H9c2 cells expressing the mitochondrial-targeted Ca²⁺ indicator, yellow cameleon 3.1. The elevation of mitochondrial Ca²⁺ concentration caused by an increase in extramitochondrial Ca²⁺ concentration was inhibited by carbonyl cyanide p -(trifluoromethoxy) phenylhydrazone (FCCP) or ruthenium red, but enhanced by CGP-37157, a mitochondrial NCX inhibitor. SEA0400 had no effect on mitochondrial Ca²⁺ under normal and ischemic conditions. Thus, the mitochondria-protective effects of SEA0400 could be explained by inhibition of plasmalemmal NCX but not mitochondrial NCX.