The cytotoxic ligand TRAIL is a promising anti-cancer agent that is entering into clinical trials. We previously identified a major subgroup of TRAIL resistant cancer cell lines with absent, or reduced DR4 expression containing a K441R polymorphism or harboring elevated levels of the caspase activation inhibitor FLIP. In the present study, we explored the use of a gene therapeutic approach utilizing p53, delivered by an adenovirus-p53 (Ad-p53) vector, which directly controls expression of the TRAIL receptor KILLER/DR5 in a panel of 8 cell lines including normal and TRAIL sensitive or resistant cancers. The functional status of the delivered p53 was monitored by detection of induced p21WAF1 expression by immunocytochemistry. In normal cells, which are TRAIL resistant, TRAIL did not reduce cell viability over and above the effect of Ad-p53 alone. All cancer cell lines were sensitive to Ad-p53 and up-regulated expression of the TRAIL receptor KILLER/DR5. TRAIL-resistant cancer cells became more sensitive to TRAIL at low Ad-p53 multiplicities of infection but TRAIL resistance was not completely overcome in one TRAIL-resistant cell line probably because of a high level of expression of FLIP. The results reveal that Ad-p53 induces the TRAIL receptor KILLER/DR5 and, like radiation or chemotherapy may effectively reverse TRAIL resistance.