Bcl-2 family proteins are central regulators of apoptosis. As the prototypic member, Bcl-2 protects various types of cells against apoptotic insults. In mammalian cells, Bcl-2 has a dual subcellular localization, in mitochondria and endoplasmic reticulum (ER). The respective roles played by mitochondrial and ER-localized Bcl-2 in apoptotic inhibition is unclear. Using Bcl-2 constructs for targeted subcellular expression, we have now determined the contributions of mitochondrial and ER-localized Bcl-2 to the anti-apoptotic effects of Bcl-2 in renal tubular cells. Wild-type Bcl-2, when expressed in renal proximal tubular cells, showed partial co-localizations with both cytochrome c and disulphide isomerase (PDI), indicating dual localizations of Bcl-2 in mitochondria and ER. In contrast, Bcl-2 constructs with mitochondria-targeting or ER-targeting sequences led to relatively restricted Bcl-2 expression in mitochondria and ER respectively. Expression of wild-type and mitochondrial Bcl-2 showed significant inhibitory effects on tubular cell apoptosis that were induced by cisplatin or ATP-depletion; however, ER-Bcl-2 was much less effective. During ATP-depletion, cytochrome c was released from mitochondria into the cytosol. This release was suppressed by wild-type and mitochondrial Bcl-2, but not by ER-Bcl-2. Consistently, wild-type and mitochondrial Bcl-2, but not ER-Bcl-2, blocked Bax activation during ATP-depletion, a critical event for mitochondrial outer membrane permeabilization and cytochrome c release. In contrast, ER-Bcl-2 protected against apoptosis during tunicamycin-induced ER stress. Collectively, the results suggest that the cytoprotective effects of Bcl-2 in different renal injury models are largely determined by it subcellular localizations.