Members of the Bcl-2 family of proteins regulate apoptosis, with some of their physiological effects mediated by their modulation of endoplasmic reticulum (ER) Ca(2+) homeostasis. Antiapoptotic Bcl-x(L) binds to the inositol trisphosphate receptor (InsP(3)R) Ca(2+) release channel to enhance Ca(2+)- and InsP(3)-dependent regulation of channel gating, resulting in reduced ER [Ca(2+)], increased oscillations of cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), and apoptosis resistance. However, it is controversial which InsP(3)R isoforms mediate these effects and whether reduced ER [Ca(2+)] or enhanced [Ca(2+)](i) signaling is most relevant for apoptosis protection. DT40 cell lines engineered to express each of the three mammalian InsP(3)R isoforms individually displayed enhanced apoptosis sensitivity compared with cells lacking InsP(3)R. In contrast, coexpression of each isoform with Bcl-x(L) conferred enhanced apoptosis resistance. In single-channel recordings of channel gating in native ER membranes, Bcl-x(L) increased the apparent sensitivity of all three InsP(3)R isoforms to subsaturating levels of InsP(3). Expression of Bcl-x(L) reduced ER [Ca(2+)] in type 3 but not type 1 or 2 InsP(3)R-expressing cells. In contrast, Bcl-x(L) enhanced spontaneous [Ca(2+)](i) signaling in all three InsP(3)R isoform-expressing cell lines. These results demonstrate a redundancy among InsP(3)R isoforms in their ability to sensitize cells to apoptotic insults and to interact with Bcl-x(L) to modulate their activities that result in enhanced apoptosis resistance. Furthermore, these data suggest that modulation of ER [Ca(2+)] is not a specific requirement for ER-dependent antiapoptotic effects of Bcl-x(L). Rather, apoptosis protection is conferred by enhanced spontaneous [Ca(2+)](i) signaling by Bcl-x(L) interaction with all isoforms of the InsP(3)R.