The protein product of the oncogene bcl-2 is a potent inhibitor of apoptotic cell death. The Bcl-2 protein has variously been reported to reside in the nuclear envelope and endoplasmic reticulum or exclusively in the inner membrane of mitochondria. We therefore undertook a detailed analysis of the intracellular distribution of Bcl-2 by immunofluorescence, immunogold electron microscopy, and subcellular fractionation in three mouse cell lines expressing a human bcl-2 transgene and measured its importation into isolated mitochondria. By these methods, the protein was localized to the nuclear envelope, the endoplasmic reticulum, and the outer mitochondrial membrane. Any proposal for the mechanism by which Bcl-2 inhibits apoptosis must therefore accommodate the fact that Bcl-2 localizes to cytoplasmic membranes facing the cytosol.