Using the yeast two-hybrid system, we have isolated a cDNA (designated BBP, for Bcl2-binding protein) for a protein (Bbp) that interacts with Bcl2. Bbp is identical to 53BP2, a partial clone of which was previously isolated in a two-hybrid screen for proteins that interact with p53. In this study, we show that specific interactions of Bbp/53BP2 with either Bcl2 or p53 require its ankyrin repeats and SH3 domain. These interactions can be reproduced in vitro with bacterially expressed fusion proteins, and competition experiments indicate that Bcl2 prevents p53 from binding to Bbp/53BP2. BBP/53BP2 mRNA is abundant in most cell lines examined, but the protein cannot be stably expressed in a variety of cell types by transfection. In transiently transfected cells, Bbp partially colocalizes with Bcl2 in the cytoplasm and results in an increased number of cells at G2/M, possibly accounting for the inability to obtain stable transfectants expressing the protein. These results demonstrate that a single protein can interact with either Bcl2 or p53 both in yeast cells and in vitro. The in vivo significance of these interactions and their potential consequences for cell cycle progression and cell death remain to be determined.