The antiapoptotic protein Bcl-2 localizes not only to mitochondria but also to the endoplasmic reticulum (ER). However, the function of Bcl-2 at the level of the ER is poorly understood. In this study, we have investigated the effects of Bcl-2 expression on Ca²⁺ storage and release by the ER. The expression of Bcl-2 decreased the amount of Ca²⁺ that could be released from intracellular stores, regardless of the mode of store depletion, the cell type, or the species from which Bcl-2 was derived. Bcl-2 also decreased cellular Ca²⁺ store content in the presence of mitochondrial inhibitors, suggesting that its effects were not mediated through mitochondrial Ca²⁺ uptake. Direct measurements with ER-targeted Ca²⁺-sensitive fluorescent “cameleon” proteins revealed that Bcl-2 decreased the free Ca²⁺ concentration within the lumen of the ER, [Ca²⁺]_ER. Analysis of the kinetics of Ca²⁺ store depletion in response to the Ca²⁺-ATPase inhibitor thapsigargin revealed that Bcl-2 increased the permeability of the ER membrane. These results suggest that Bcl-2 decreases the free Ca²⁺ concentration within the ER lumen by increasing the Ca²⁺ permeability of the ER membrane. The increased ER Ca²⁺ permeability conferred by Bcl-2 would be compatible with an ion channel function of Bcl-2 at the level of the ER membrane.