In order to study the mechanisms by which p53 function is regulated, human wild-type p53 cDNA was cloned into a vaccinia virus vector and the expressed p53 protein was used to investigate binding of the p53 by cellular proteins from a cDNA expression library from human liver. One protein that bound wild-type p53 had > 99% homology with DNA topoisomerase IIb. p53 protein was coimmunoprecipitated from topoisomerase II-rich cell lysates (but not from topoisomerase II-deficient cell lysates) by an antibody to topoisomerase IIa and IIb. This binding was shown to occur without a dsDNA intermediary. Hepatocellular carcinomas (HCCs) and adjacent nontumorous liver tissues from ten patients were studied to determine the level of expression of topoisomerase II and p53. Overexpressed topoisomerase II proteins were detected by western blot in six of ten HCCs (60%), including several in which presumed wild-type p53 was detected by immunohistochemistry. No topoisomerase II expression was detectable in the ten nontumorous liver tissues from the same patients or in a sample of normal human liver.