BAX and BAK are "multidomain" proapoptotic proteins that initiate mitochondrial dysfunction but also localize to the endoplasmic reticulum (ER). Mouse embryonic fibroblasts deficient for BAX and BAK (DKO cells) were found to have a reduced resting concentration of calcium in the ER ([Ca2+]er) that results in decreased uptake of Ca2+ by mitochondria after Ca2+ release from the ER. Expression of SERCA (sarcoplasmic-endoplasmic reticulum Ca2+ adenosine triphosphatase) corrected [Ca2+]er and mitochondrial Ca2+ uptake in DKO cells, restoring apoptotic death in response to agents that release Ca2+ from intracellular stores (such as arachidonic acid, C2-ceramide, and oxidative stress). In contrast, targeting of BAX to mitochondria selectively restored apoptosis to "BH3-only" signals. A third set of stimuli, including many intrinsic signals, required both ER-released Ca2+ and the presence of mitochondrial BAX or BAK to fully restore apoptosis. Thus, BAX and BAK operate in both the ER and mitochondria as an essential gateway for selected apoptotic signals.