Although recent studies focused on the contribution of mitochondrial Ca²⁺ for the mechanisms of ischemia/reperfusion injury, the regulation of mitochondrial Ca²⁺ under the pathophysiological condition remains largely unclear. By using saponin-permeabilized rat myocytes, we measured mitochondrial membrane potential (_m) and mitochondrial Ca²⁺ concentration ([Ca²⁺]_m) at the physiological range of cytosolic Ca²⁺ concentration ([Ca²⁺]_c= 300 nM), and investigated the regulation of [Ca²⁺]_m both in the normal and dissipated _m When _m was partially depolarized by FCCP (0.01 - 0.1 µM), there were dose-dependent decreases in [Ca²⁺]_m. When the complete _m dissipation was achieved by FCCP (0.3 - 1 µM), [Ca²⁺]_m remained at the half of control level, in spite of no Ca²⁺ influx via the Ca²⁺ uniporter. The _mdissipation by FCCP accelerated calcein leakage from mitochondria in a cyclosporin A (CsA)-sensitive manner, indicating that the _m dissipation opened mitochondrial permeability transition pore (mPTP). While the inhibition of mPTP by CsA caused further [Ca²⁺]_m reduction after FCCP, the inhibition of mitochondrial Na⁺/Ca²⁺ exchange (mitoNCX) by a Na⁺-free solution abolished the [Ca²⁺]_m reduction after FCCP. The cytosolic Na⁺ concentrations, which give a half-maximal activity of mitoNCX, were 3.6 mM in the normal _m and 7.6 mM in the _m dissipation. We conclude that 1) the mitochondrial Ca²⁺ uniporter accumulates Ca²⁺ _m dependently at the physiological range of [Ca²⁺]_c, 2) the _m dissipation opens mPTP, resulting in a Ca²⁺ influx into mitochondria, 3) although the activity of mitoNCX is impaired, mitoNCX extrudes Ca²⁺ from matrix even after the _m, dissipation.