Calcium is a key ion and is known to mediate signaling pathways between cytosol and mitochondria and modulate mitochondrial energy metabolism. To gain a quantitative, biophysical understanding of mitochondrial Ca²⁺ regulation, we developed a thermodynamically-balanced model of mitochondrial Ca²⁺ handling and bioenergetics by integrating kinetic models of mitochondrial Ca²⁺ uniporter (CU), Na⁺/Ca²⁺ exchanger (NCE), and Na⁺/H⁺ exchanger (NHE) into an existing computational model of mitochondrial oxidative phosphorylation (Beard, PLoS Comp Biol 1:e36, 2005). Kinetic flux expressions for the CU, NCE and NHE were developed and individually parameterized based on independent data sets on flux rates measured in purified mitochondria. While available data supports a wide range of possible values for the overall activity of the CU in cardiac and liver mitochondria, even at the highest estimated values, the Ca²⁺ current through the CU does not have a significant effect on mitochondrial membrane potential. This integrated model was then used to analyze additional data on the dynamics and steady-states of mitochondrial Ca²⁺ governed by mitochondrial CU and NCE. Our analysis of the data on the time course of matrix free [Ca²⁺] in respiring mitochondria purified from rabbit heart with addition of different levels of Na⁺ to the external buffer medium (with the CU blocked) with two separate models—one with a 2:1 stoichiometry and the other with a 3:1 stoichiometry for the NCE—supports the hypothesis that the NCE is electrogenic with a stoichiometry of 3:1. This hypothesis was further tested by simulating an additional independent data set on the steady state variations of matrix free [Ca²⁺] with respect to the variations in external free [Ca²⁺] in purified respiring mitochondria from rat heart to show that only the 3:1 stoichiometry model predictions are consistent with the data. Based on these analyses, it is concluded that the mitochondrial NCE is electrogenic with a stoichiometry of 3:1.