The efficacy of adenovirus (ADV)-mediated gene therapy to treat brain tumors was tested in a syngeneic glioma model. Tumor cells were transduced in situ with a replication-defective ADV carrying the herpes simplex virus thymidine kinase (HSV-tk) gene controlled by the Rous sarcoma virus promoter. Expression of the HSV-tk gene enables the transduced cell to convert the drug ganciclovir to a form that is cytotoxic to dividing cells. Tumors were generated in Fischer 344 rats by stereotaxic implantation of 9L gliosarcoma cells into the caudate nucleus. Eight days later, the tumors were injected either with the ADV carrying the HSV-tk (ADV-tk) gene or a control ADV vector containing the P-galactosidase (ADV-Pgal) gene and the rats were treated with either ganciclovir or saline. Tumor size was measured 20 days after implantation of 9L cells or at death. Rats treated with ADV-Pgal and ganciclovir or with ADV-tk and saline had large tumors. No tumors were detected in animals treated with ADV-tk and with ganciclovir at doses 280 mg/kg. An infiltrate of macrophages and lymphocytes at the injection site in animals treated with ADV-tk and ganciclovir indicated an active local immune reaction. In survival studies, all animals treated with ADV-tk and ganciclovir have remained alive longer than 80 and up to 120 days after tumor induction whereas all untreated animals died by 22 days. These results demonstrate that ADV-mediated transfer of HSV-tk to glioma cells in vivo confers sensitivity to ganciclovir, and represents a potential method of treatment of brain tumors.