Viral vector-mediated transfer of chemosensitization genes represents a promising new approach to the treatment of cancer. Previous reports have demonstrated that transfection of the bacterial cytosine deaminase (cd) gene into mammalian cells can sensitize them to the otherwise nontoxic nucleoside, 5-fluorocytosine (5-FC). We now report that a replication-deficient adenovirus vector that transduces the cd gene (Ad.CMV-cd) highly sensitizes 9L gliosarcoma cells to 5-FC, and that gene transduction is associated with a potent bystander effect that is not dependent on direct cell-to-cell contact. Stereotactic injection of Ad.CMV-cd into established rat gliomas, followed by systemic administration of 5-FC in vivo, results in prolongation of survival.