Previous studies have demonstrated that the conditionally replicative adenovirus Ad5Delta24 is a powerful cytolytic agent against glioma selectively affecting cells with a defective p16/Rb/E2F pathway. The p53 protein is also known to be an apoptotic factor for glioma cells. In this study, we examined the simultaneous delivery of the combination of exogenous p53 and Ad5Delta24 adenovirus in glioma cells. Infecting cells with low doses of adenovirus p53 and Ad5Delta24 resulted in an additive effect on cell death. The cell death induced by both agents was independent of the p53 status of cells. Flow cytometry revealed that the potent anti-tumor effect induced by the mixture of Ad5CMV-p53 and Ad5Delta24 adenoviruses was due to a combination of apoptosis and cell lysis. Our results indicate that Ad5CMV-p53 enhances the oncolytic effect of the Ad5Delta24 adenovirus, and the combination of adenovirus Ad5Delta24 and Ad5CMV-p53 may thus be a potential therapeutic tool for gliomas.