The cardioprotective effects of SEA0400, a novel Na+–Ca2+ exchanger inhibitor, were examined in isolated guinea pig myocardial tissue and ventricular myocytes. In a coronary-perfused right ventricular tissue preparation, SEA0400 had no cardiosuppressive effect during normoxia and experimental ischemia, but enhanced the recovery of contractile force during reperfusion. SEA0400 had no effect on tissue ATP content during normoxia, but attenuated its decrease during ischemia. Treatment of ventricular myocytes with an ischemia mimetic solution (high K+, glucose free, pH 6.0, gassed with N2) resulted in the depolarization of the mitochondrial membrane potential and an increase in cytoplasmic and mitochondrial Ca2+ concentration, which had a similar time course. SEA0400 significantly delayed these changes. These results suggest that SEA0400 maintains mitochondrial function and tissue ATP content during ischemia through the inhibition of cytoplasmic and mitochondrial Ca2+ overload.