Inositol 1,4,5-trisphosphate (InsP₃) receptors (InsP₃Rs) are channels responsible for calcium release from the endoplasmic reticulum (ER). We show that the anti-apoptotic protein Bcl-2 (either wild type or selectively localized to the ER) significantly inhibited InsP₃-mediated calcium release and elevation of cytosolic calcium in WEHI7.2 T cells. This inhibition was due to an effect of Bcl-2 at the level of InsP₃Rs because responses to both anti-CD3 antibody and a cell-permeant InsP₃ ester were decreased. Bcl-2 inhibited the extent of calcium release from the ER of permeabilized WEHI7.2 cells, even at saturating concentrations of InsP₃, without decreasing luminal calcium concentration. Furthermore, Bcl-2 reduced the open probability of purified InsP₃Rs reconstituted into lipid bilayers. Bcl-2 and InsP₃Rs were detected together in macromolecular complexes by coimmunoprecipitation and blue native gel electrophoresis. We suggest that this functional interaction of Bcl-2 with InsP₃Rs inhibits InsP₃R activation and thereby regulates InsP₃-induced calcium release from the ER.