Members of the Bcl-2 protein family play a central role in the regulation of apoptosis. An interaction between anti-apoptotic Bcl-x(L) and the endoplasmic reticulum (ER)-localized inositol trisphosphate receptor Ca(2+) release channel (InsP(3)R) enables Bcl-x(L) to be fully efficacious as an anti-apoptotic mediator (White, C., Li, C., Yang, J., Petrenko, N. B., Madesh, M., Thompson, C. B., and Foskett, J. K. (2005) Nat. Cell Biol. 7, 1021-1028). Physiologically, Bcl-x(L) binds to the InsP(3)R to enhance its gating and Ca(2+) signaling. Here we have discovered that structurally related proteins Bcl-2 and Mcl-1 function similarly. Bcl-2, Mcl-1 and Bcl-x(L) bind with comparable affinity to the carboxyl termini of all three mammalian InsP(3)R isoforms with important functional consequences. Stable expression of Bcl-2 or Mcl-1 lowered ER Ca(2+) content and enhanced the rate of InsP(3)-mediated Ca(2+) release in response to submaximal InsP(3) stimulation in permeabilized wild-type DT40 cells but not in cells lacking InsP(3)R. In addition, expression of either Bcl-2 or Mcl-1 enhanced spontaneous InsP(3)R-dependent Ca(2+) oscillations and spiking in intact cells in the absence of agonist stimulation. Bcl-2- and Mcl-1-mediated protection from apoptosis induced by staurosporine or etoposide was enhanced in cells expressing InsP(3)R, demonstrating that their interactions with InsP(3)R enable Bcl-2 and Mcl-1 to be fully efficacious anti-apoptotic mediators. Our data suggest a molecular mechanism that is shared by several anti-apoptotic Bcl-2 proteins that provides apoptosis resistance by direct interactions at the ER with the InsP(3)R that impinges on cellular Ca(2+) homeostasis.