The p53AIP1 gene, which we recently identified as a novel p53-target, mediates p53-dependent apoptosis. We evaluated the effects of adenovirus-mediated introduction of p53AIP1 (Ad-p53AIP1) on 30 human cancer-cell lines in vitro, and two cell lines in vivo, in comparison with the effects of p53 (Ad-p53). In 20 of the 30 cell lines, p53AIP1-induced apoptosis was observed, and in 12 of these p53AIP1-sensitive cancer cell lines, the apoptotic effects of p53AIP1 were greater than those of p53 itself. Cancers with wild-type p53, which were thought to be p53-resistant, were likely to be sensitive to p53AIP1-induced apoptosis. p53-resistant cancers such as LS174T (p53 +/+) and A549 (p53 +/+), in which no increase of p53AIP1 mRNA expression was observed when Ad-p53 was introduced, were killed effectively by Ad-p53AIP1. Furthermore, co-introduction of p53 and p53AIP1 had a synergistic effect on the induction of apoptosis, regardless of p53 status. Finally, adenovirus-mediated introduction of p53AIP1 suppressed tumor growth in vivo. These results suggested that p53AIP1 gene transfer might become a new strategy for the treatment of p53-resistant cancers.