We have previously shown that the introduction of human recombinant wild-type p53 mediated by an adenoviral vector (Ad5CMV-p53), either alone or delivered in combination with ionizing radiation, was cytotoxic to two nasopharyngeal carcinoma (NPC) cell lines. To further explore the potential therapeutic role for gene therapy, the combination of Ad5CMV-p53 and cisplatin was examined in two NPC cell lines, CNE-1 and C666-1. The C666-1 cells are particularly relevant because they express Epstein-Barr virus latent gene products analogous to human NPC in situ. Cells were infected with 5 pfu/cell of Ad5CMV-p53 or Ad5CMV-beta-gal, followed by exposure to increasing doses of cisplatin. Clonogenic and MTT assays were used to assess the sensitivity of cells to these treatments, and apoptosis was also quantified. The combination of Ad5CMV-p53 and cisplatin resulted in approximately 25% greater cytotoxicity compared to that observed with cisplatin alone in either cell line. Apoptosis was induced in approximately 50% of cells following administration of both Ad5CMV-p53 and cisplatin, but was induced in considerably fewer cells following either treatment alone. The two modalities appeared to interact in an additive manner. Ad5CMV-p53 gene therapy resulted in the expression of biologically active p53 protein, shown by induction of p21(WAF1/CIP1). Cisplatin treatment showed little effect on either p53 or p21(WAF1/CIP1) expression. Therefore, both p53 gene therapy and cisplatin chemotherapy demonstrated cytotoxicity mediated by apoptosis despite the presence of EBV gene products in the C666-1 cells, but it appears that the two modalities induce cytotoxicity by independent pathways.