Our previous work in cultured cells has shown that the maintenance of mitochondrial Ca²⁺homeostasis is essential for cell survival, and that the anti-apoptotic protein Bcl-2 is able to maintain a threshold level of mitochondrial Ca²⁺by the inhibition of permeability transition. To test whether Bcl-2 also affects the mitochondrial Na⁺–Ca²⁺exchange (NCE), a major efflux pathway for mitochondrial Ca²⁺, studies using transgenic mice that overexpress Bcl-2 in the heart have been performed. NCE activity was determined as the Na⁺-dependent Ca²⁺efflux in the isolated mitochondria. Overexpression of Bcl-2 led to a significant reduction of NCE activity as well as increased resistance to permeability transition in the mitochondria of transgenic heart. This was accompanied by increased matrix Ca²⁺level, enhanced formation of NADH and enhanced oxidation of pyruvate, an NAD⁺-linked substrate. Furthermore, there was induction of cellular Ca²⁺transport proteins including the Na⁺–Ca²⁺exchanger of the sarcolemma (NCX). Bcl-2 not only stimulates NCX expression in the sarcolemma but also attenuates the Na⁺–Ca²⁺exchange in the mitochondria. These results are consistent with the protection by Bcl-2 against apoptosis in heart following ischemia/reperfusion.