Human Bcl‐2 is located in multiple intracellular membranes when expressed in MDCK and Rat‐1/myc cells. We restricted expression to the endoplasmic reticulum or mitochondria by exchanging the Bcl‐2 carboxy‐terminal insertion sequence for an equivalent sequence from cytochrome b5 or ActA, respectively. MDCK cells are protected from serum deprivation‐induced apoptosis by both wild‐type Bcl‐2 and the mutant targeted to mitochondria but not by the mutant targeted to endoplasmic reticulum. In contrast, when expressed in Rat‐1/myc cells, the Bcl‐2 mutant located at the endoplasmic reticulum is more effective than that targeted to mitochondria. In MDCK cells both mutants bind Bax as effectively as wild‐type, demonstrating that Bax binding is not sufficient to prevent apoptosis.