Inositol 1,4,5-trisphosphate (InsP3) induced release of calcium ions from the endoplasmic reticulum (ER) can induce apoptosis directly by activating cell extrinsic and intrinsic death pathways. Here we report that the anti-apoptotic protein Bcl-2, which resides on the ER membrane, inhibits InsP3-mediated calcium release. Cytoplasmic calcium was measured in the WEHI7.2 T cell line with the calcium indicator Fura-2 (AM), both fluorometrically and by single cell imaging. Bcl-2 markedly suppressed cytoplasmic calcium elevation induced by either antibody to the CD3 component of the T cell receptor or D-myo InsP3BM, a cell permeant InsP3 ester. This action of Bcl-2 was not due to a decrease in the thapsigargin-releasable ER calcium pool, nor was it due to a reduction in lumenal free calcium concentration, measured in digitonin-permeabilized cells with the low affinity dye Fura-2FF (AM). Instead, we found that Bcl-2 suppressed the activity of InsP3 receptors without altering their level of expression. The mechanism involved an interaction of Bcl-2 with InsP3 receptors, demonstrated by co-immunoprecipitation. This interaction decreased both the state of InsP3 receptor phosphorylation, measured by 32P-labeling, and the affinity of InsP3 receptors for InsP3, measured by a competitive binding assay. In summary, these findings indicate that Bcl-2 is a novel InsP3-receptor interacting protein that suppresses InsP3-linked apoptotic signals by reducing the responsiveness of InsP3 receptors to InsP3.