In mitochondria-dependent apoptosis in mammalian cells, the downstream signals (such as the release of cytochrome c) and the activation of caspases family were well studied. However, the upstream signal of mitochondria is still not clear. It is known that the Bcl-2 family proteins play important roles in controlling the permeabilization of the mitochondrial outer membrane. Alternatively, there is evidence suggesting that Ca²⁺ signal is also involved in regulating the apoptotic process. Recently, it was found that Bcl-2 family proteins can affect the apoptotic process by modifying Ca²⁺ released from the ER (1). The objective of this work is to understand the cross talk between the Bcl-2 signaling pathway and the Ca²⁺ signaling pathway. First, we examined whether mitochondrial Ca²⁺ plays a role in the apoptotic process. Previously, we found that cytosolic Ca²⁺ signal was upstream of cytochrome c release during apoptosis in HeLa cells (2). Using Rhod-2-AM as a mitochondrial Ca²⁺ indicator, we observed a bi-phase mitochondrial Ca²⁺ elevation during the apoptotic process. In HeLa cells undergoing the early stage of UV-induced apoptosis, we observed mitochondrial Ca²⁺ oscillation that was synchronized with cytosolic Ca²⁺ elevation. At a later apoptotic stage, a broader mitochondrial Ca²⁺ elevation was observed before mitochondrial membrane potential decrease. Second, we examined how the bcl-2 family proteins affect the Ca²⁺ mobilization from ER to mitochondria. We fused various bcl-2 proteins with different GFP and dsRed mutants and examined their overexpression effects on Ca²⁺ signal. We found that Bcl-2 and Bcl-xL can affect the ER Ca²⁺ homeostasis, while another mitochondria-localized Bcl-2 family protein, Mcl-1, can reduce mitochondrial Ca²⁺ uptake. Third, we investigated the functional role of IP3 receptors (IP3Rs) in apoptosis. We found that IR3Rs inhibitors (2-APB, Heparin) in general were anti-apoptotic, while an IP3R binding protein, RACK1, which can sensitize IP3Rs, promoted UV-induced apoptosis. These findings support the hypothesis that suppressing the Ca²⁺ mobilization between ER and mitochondria has an anti-apoptotic effect. We are currently conducting further experiments to study the interaction between IP3Rs and Bcl-2 family proteins.