Bax Inhibitor-1 (BI-1) is an evolutionarily conserved six-transmembrane domain endoplasmic reticulum (ER)-localized protein that protects against ER stress-induced apoptotic cell death. This function is closely connected to its ability to lower steady-state ER Ca 2+ levels. Recently, we elucidated BI-1's Ca 2+ -channel pore in the C-terminal part of the protein and identified the critical amino acids of its pore. Based on these insights, a Ca 2+ -channel pore-dead mutant BI-1 (BI-1 D213R ) was developed. We determined whether BI-1 behaves as a bona fide H + /Ca 2+ antiporter or as an ER Ca 2+ -leak channel by investigating the effect of pH on unidirectional Ca 2+ -efflux rates. At pH 6.8, wild-type BI-1 expression in BI-1 −/− cells increased the ER Ca 2+ -leak rate, correlating with its localization in the ER compartment. In contrast, BI-1 D231R expression in BI-1 −/− , despite its ER localization, did not increase the ER Ca 2+ -leak rate. However, at pH<6.8, the BI-1-mediated ER Ca 2+ leak was blocked. Finally, a peptide representing the Ca 2+ -channel pore of BI-1 promoting Ca 2+ flux from the ER was used. Lowering the pH from 6.8 to 6.0 completely abolished the ability of the BI-1 peptide to mediate Ca 2+ flux from the ER. We propose that this pH dependence is due to two aspartic acid residues critical for the function of the Ca 2+ -channel pore and located in the ER membrane-dipping domain, which facilitates the protonation of these residues.