The adenomatous polyposis coli or beta-catenin genes are frequently mutated in colorectal cancer cells, resulting in oncogenic activation of beta-catenin signaling. We tried to establish in vitro and in vivo models for selectively killing human cancer cells with an activated beta-catenin/T-cell factor (Tcf) pathway. We used a recombinant adenovirus that carries a lethal gene [p53-up-regulated modulator of apoptosis (PUMA)] under the control of a beta-catenin/Tcf-responsive promoter (AdTOP-PUMA) to selectively target human colorectal cancer cells (SW480, HCT116, DLD-1, and LS174T), hepatocellular carcinoma (HepG2), and gastric cancer cells (AGS) in which the beta-catenin/Tcf pathway is activated, and compared its efficiency in killing cancer cells in which this pathway is inactive or only weakly active. AdFOP-PUMA, carrying a mutant Tcf-binding site, was used as control virus. The combined effect of AdTOP-PUMA with several chemotherapeutic agents (5-florouracil, doxorubicin, and paclitaxel) was also evaluated. The effect of AdTOP-PUMA on colorectal cancer cells was also examined in nude mice: SW480 cells were infected with the AdTOP-PUMA and AdFOP-PUMA, and then inoculated s.c. into nude mice. The TOP-PUMA adenovirus inhibited cell growth in a dose-dependent fashion, depending on the signaling activity of beta-catenin. The growth of cells displaying high levels of active beta-catenin/Tcf signaling was inhibited after infection with AdTOP-PUMA, whereas that of cells with low levels of beta-catenin signaling was not. Growth inhibition was associated with induction of apoptosis. Chemotherapy synergistically enhanced the effect of AdTOP-PUMA. A combination of the adenovirus system with standard therapy may improve the efficacy and reduce the toxicity of therapy in humans.