Mitochondrial Ca2+ regulation is closely related to cellular function including energy production and cell viability. Intramitochondrial Ca2+ overload is eventually destined to cellular death, therefore, the modulation of the mitochondrial Ca2+ uptake is important to regulate cellular function. In this study, we would like to see the effect of inorganic phosphate (Pi), pH, adenosine phosphates and Mg2+ on mitochondrial Ca2+ uptake kinetics with newly developed quantitative method. We enzymatically isolated single ventricular myocytes of rat. All procedures were accorded with national legislation. Buthanedione monoxime was present to prevent contracture. We measured NADH, mitochondrial Ca2+ with Fura-2FF and mitochondrial membrane potential (ψm) with tetramethylrhodamine ethyl ester, simultaneously. To maintain mitochondrial membrane potential, 5 mM of pyruvate and malate were present at all times. The Ca2+ uptake rate was increased as extramitochondrial Ca2+ was increased in a dose dependent manner. As intramitochondrial Ca2+ increased, NADH and mitochondrial membrane potential was decreased. The half-activating Ca2+ concentration (Kh) was about 0.33 μM. In the presence of Pi,Ca2+ uptake was not occurred properly and mitochondrial membrane potential was rapidly depolarized. These phenomena were prevented by ATP and the Kh was 1.79 μM in the presence of ATP and Pi. The Kh was shifted to the right, compared to the control Kh. ADP did not shift the Kh. Mg2+ markedly shifted the Ca2+ sensitivity to the right and the Kh value was about 1.83 μM. In addition, the maximal uptake rate also decreased. Cytosolic acidification to pH 6.0 also shifted Kh to the right and the Kh was about 1.56 μM. From the above results, ATP, Mg2+ and acidic pH decreased the cytosolic Ca2+ sensitivity of mitochondrial Ca2+ influx and those modulations must be important in diverse pathologic conditions.