p16 is an important regulator of the cell cycle at the G(1) phase. Frequent aberration of p16 in nasopharyngeal carcinoma (NPC) suggests a role for this tumor suppressor gene in disease development. p16 gene transfer has been demonstrated to be effective in various human cancer models, including breast, lung, and prostate, causing cell cycle arrest, apoptosis, and tumor growth delay. We investigated the potential of adenoviral-mediated p16 therapy, in combination with ionizing radiation (RT), in two distinct NPC models. Two deltaE1 adenoviral vectors were employed: one carrying the human p16 gene (adv.p16), and the other a beta-galactosidase reporter gene (adv.beta-gal), both driven by the cytomegalovirus (CMV) promoter. Two NPC cell lines with differential endogenous p16 expression, CNE-1 (low) and CNE-2Z (high), were evaluated for protein expression, cytotoxicity, cell cycle analysis, apoptosis, and senescence. The CNE-1 cells were exquisitely sensitive to adv.p16, with 0.1% survival level after gene therapy [25 plaque-forming unit (pfu)/cell], which further decreased to 0.01% with the addition of RT (2 Gy). This reduction in survival was effected through necrosis, G1 arrest, and senescence. In contrast, CNE-2Z cells were resistant to adv.p16 gene transfer, with 75% surviving at an equivalent viral dose. This differential sensitivity was recapitulated in vivo in that adv.p16-treated CNE-1 cells formed no tumors in severe-combined-immunodeficiency (SCID) mice, followed for over 100 days. In contrast, tumor formation was detected 40 days after implantation of adv.p16-treated CNE-2Z cells. In conclusion, adv.p16 gene transfer appears to be highly effective against NPC that lack functional p16, which is the situation in the majority of NPC patients.