Replication-competent adenoviruses could provide an efficient method for delivering therapeutic genes to tumors. The most promising strategies among adenovirus-based oncolytic systems are designed to exploit free E2F-1 activity in cancer cells, which in the absence of pRb activates transcription and regulates the expression of genes involved in differentiation, proliferation, and apoptosis. We previously developed Δ24, an E1A-mutant, conditionally replicative oncolytic adenovirus. Here, we examine the ability of a second-generation Δ24 (Δ24-hyCD) engineered to express a humanized form of the Saccharomyces cerevisiae cytosine deaminase gene (hyCD). Real-time quantitative PCR, Western blotting, thin-layer chromatography, and radioisotope quantitative enzymatic assays confirmed the production of a catalytically active hyCD enzyme in the setting of an oncolytic infection in vitro; other experiments assessing local production of 5-fluorouracil and a concomitant bystander effect showed improved cytotoxicity. The IC50 dose of 5-fluorocytosine (5-FC) required for a complete cytopathic effect by the Δ24-hyCD virus was fivefold lower than with Δ24 alone in U251MG and U87MG malignant glioma (MG) cell lines. Intratumoral treatment of mice bearing intracranial U87MG xenografts with Δ24-hyCD + 5-FC significantly improved survival, confirming that Δ24-hyCD with 5-FC is a more efficient anticancer tool than Δ24 alone. Histopathologically, Δ24-hyCD replication was accompanied by progressively augmented oncolysis and drug-induced necrosis. These findings demonstrate that Δ24-hyCD with concomitant systemic 5-FC is a significant improvement over the earlier Δ24 oncolytic tumor-selective strategy for therapy of experimental gliomas. © 2005 Nature Publishing Group All rights reserved.