To clarify the property of mitochondrial Na+/Ca2+ exchange in situ, we measured mitochondrial Ca2+ using Rhod-2 in permeabilized rat ventricular myocytes. Cytoplasmic 300 nM Ca2+ (Ca2+(c)) augmented the Rhod-2 intensity by approximately ninefolds without cytoplasmic Na+ (Na+(c)). Increasing Na+(c) attenuated the maximum level of Rhod-2 fluorescence, probably due to the activation of forward mode of mitochondrial Na+/Ca2+ exchange. The Rhod-2 intensity decayed upon removing Ca2+(c). The decay was dependent on Na+(c) (K(1/2) = approximately 1 mM) and largely abolished by an inhibitor of mitochondrial Na+/Ca2+ exchange, CGP-37157. It was suggested that Na+ binding to the mitochondrial Na+/Ca2+ exchange is saturated in the physiological concentration of Na+(c).