We examined the effects of SEA0400 and CGP-37157 on the plasmalemmal Na⁺–Ca²⁺ exchanger (NCX) and mitochondrial NCX using H9c2 cardiomyocytes loaded with Ca²⁺-sensitive fluorescent probes. The plasmalemmal NCX activity, which was measured as the increase in cytoplasmic Ca²⁺ concentration after application of low Na⁺ extracellular solution, was inhibited by SEA0400 but not by CGP-37157. The mitochondrial NCX activity, which was measured in permeabilized H9c2 cells as the decrease in mitochondrial Ca²⁺ concentration after application of Ca²⁺-free extramitochondrial solution, was inhibited by CGP-37157 but not by SEA0400. These results indicate that SEA0400 and CGP-37157 act as selective inhibitors towards plasmalemmal and mitochondrial NCX, respectively, and provide pharmacological evidence that the plasmalemmal and mitochondrial NCX are distinct molecular entities.