The p53 binding protein, termed p53BP2, was identified as a protein interacting with protein phosphatase 1 (PP1) in the yeast two hybrid system. The interaction was confirmed by co-immunoprecipitation of p53BP2 with epitope-tagged PP1 in vitro. The p53BP2-PP1 complex was stable to NaCl at concentrations which dissociate the p53-p53BP2 complex, and the binding of PP1 and p53 to p53BP2 was mutually exclusive. The region required for interaction with PP1 was shown to be contained within amino acids 297-431 of p53BP2, which includes two ankyrin repeats. The phosphorylase phosphatase activity of PP1 was inhibited by p53BP2 at nanomolar concentrations. These results suggest that PP1 may be involved in dephosphorylation and regulation of p53 through interaction with p53BP2.