p53 exerts important physiological functions in cell-cycle control, gene regulation, cell differentiation, apoptosis and tumor suppression by interacting with many cellular proteins. Using the yeast two-hybrid system, we screened a HeLa cDNA library and identified a novel gene encoding a p53-binding protein (p53BP3). The full-length cDNA of p53BP3 was isolated from a HeLaλgt10 cDNA library. This predicted protein was composed of 815 amino acids. Sequence analysis indicated that p53BP3 contained two bipartite nuclear localization signals and was confirmed to be a nuclear protein. FISH mapping results showed that this novel gene was located at human chromosome 12, region p11.2-p12.1. Northern blot analysis suggested that p53BP3 was broadly expressed in human tissues. A further study showed that p53BP3 had a homologue in mouse.