The role of mitochondrial Na+-Ca2+ exchange is crucial for the efflux of mitochondrial Ca2+ which regulates the mitochondrial metabolism. The electrogenicity of mitochondrial Na+-Ca2+ exchange has been controversial and no membrane current through the Na+-Ca2+ exchanger has been reported. We investigated the electrogenicity of mitochondrial Na+-Ca2+ exchange with voltage clamp experiments and succeeded in measuring the mitochondrial Na+-Ca2+ exchange currents for the first time. Mitochondria were isolated from mouse heart and were exposed to a hypotonic solution to form mitoplasts with a diameter of 3 - 5 μm, which were then used for whole mitoplast patch clamp experiments. Under conditions that K+ and Cl- currents, and Ca2+ uniporter current were inhibited, extra-mitochondrial application of 12.5 - 50 mM Na+ induced inward currents with 1 μM Ca2+ in the pipette. The inward current was diminished without Ca2+ in the pipette, and was augmented with 10 μM Ca2+. With 100 mM Na+ in the pipette, extra-mitochondrial application of 10 μM-1 mM Ca2+ induced outward currents. Both the Na+-induced inward and the Ca2+-induced outward currents were largely inhibited by 2 μM CGP-37157, a mitochondrial Na+-Ca2+ exchange blocker. On the contrary Na+-induced inward current was not inhibited by 1 μM of SEA0400, a plasma membrane NCX1 blocker. These results demonstrated the direct evidence that the mitochondrial Na+-Ca2+ exchange is electrogenic.