In this study, Bax inhibitor-1 (BI-1) overexpression reduces the ER pool of Ca(2+) released by thapsigargin. Cells overexpressing BI-1 also showed lower intracellular Ca(2+) release induced by the Ca(2+) ionophore ionomycin as well as agonists of ryanodine receptors and inositol trisphosphate receptors. In contrast, cells expressing carboxyl-terminal deleted BI-1 (CDelta-BI-1 cells) displayed normal intracellular Ca(2+) mobilization. Basal Ca(2+) release rates from the ER were higher in BI-1-overexpressing cells than in control or CDelta-BI-1 cells. We determined that the carboxyl-terminal cytosolic region of BI-1 contains a lysine-rich motif (EKDKKKEKK) resembling the pH-sensing domains of ion channels. Acidic conditions triggered more extensive Ca(2+) release from ER microsomes from BI-1-overexpressing cells and BI-1-reconstituted liposomes. Acidic conditions also induced BI-1 protein oligomerization. Interestingly subjecting BI-1-overexpressing cells to acidic conditions induced more Bax recruitment to mitochondria, more cytochrome c release from mitochondria, and more cell death. These findings suggest that BI-1 increases Ca(2+) leak rates from the ER through a mechanism that is dependent on pH and on the carboxyl-terminal cytosolic region of the BI-1 protein. The findings also reveal a cell death-promoting phenotype for BI-1 that is manifested under low pH conditions.