p14(ARF), a product of the INK4A/ARF locus, induces p53 upregulation by neutralizing the effects of MDM2, a transcriptional target of p53 that antagonizes its function. Here we report that adenovirus-mediated p14(ARF) gene transfer leads to the accumulation of ectopically transduced p53 and to apoptosis in human cancer cells. We constructed an adenoviral vector expressing p14(ARF) (Ad-ARF) and examined its synergistic effect with p53-expressing adenovirus (Ad5CMV-p53 or Ad-p53) in human lung and esophageal cancer cells. Simultaneous Ad-ARF and Ad-p53 infection increased p53 protein levels not only in a wild-type p53-expressing cell line, but also in cell lines with deleted p53. This resulted in a significant in vitro cytotoxicity compared with Ad-p53 infection alone. Coinfection of Ad-ARF and Ad-p53 also resulted in an increase in expression of p53-inducible genes, including p21(WAF-1/Cip1), p53R2, and Noxa. In addition, the growth of human lung cancer tumors subcutaneously implanted into nu/nu mice was inhibited significantly by intratumoral injection with Ad-ARF and Ad-p53. Our data demonstrate that overexpression of ectopic p14(ARF) may render cells more sensitive to p53-mediated apoptosis, an outcome that has important implications for the treatment of human cancers.