PRPK (p53-related protein kinase) has been reported as a novel protein kinase which binds to the tumor suppressor protein p53 and induces phosphorylation of p53 at Ser 15. To identify novel binding partners of PRPK, we performed a yeast two-hybrid screening and isolated an expressed sequence tag CGI-121 by which a 20-kDa protein was encoded. We demonstrated the protein-protein interaction of CGI-121 with PRPK in vivo and in vitro. The protein expression of CGI-121 was observed in many cell lines and was immunocytochemically identified in both the nucleus and cytosol. Although PRPK interacted with both CGI-121 and p53, several attempts to demonstrate an association between CGI-121 and p53 were unsuccessful. In addition, coprecipitation of p53 using recombinant PRPK was inhibited by adding recombinant CGI-121 in vitro, suggesting that CGI-121 could act as a potent inhibitor of the binding of PRPK to p53.