A major obstacle to the successful application of suicide gene therapy strategies that rely on in situ transduction of tumor cells is the poor distribution of the vector throughout the tumor mass. To address this problem, we evaluated the use of Ad.TK^RC, an E1b M_r 55,000 deleted replicating adenoviral vector engineered to express the herpes simplex virus type 1 thymidine kinase gene (HSV-tk) in combination with ganciclovir (GCV) as a treatment for human colon cancer xenografts in nude mice. We compared the efficacy of this system with that of a standard replication-deficient adenovirus expressing HSV-tk (Ad.TK) in mice bearing LS180 tumors. In this system, Ad.TK^RC alone was as effective as a traditional Ad.TK vector in combination with GCV. The addition of GCV significantly enhanced the antitumor effect of Ad.TK^RC. Furthermore, we demonstrated that the survival of HT-29 human colon cancer xenografted mice treated with Ad.TK^RC and GCV was prolonged compared with Ad.TK^RC alone or with administration of a single cycle of topotecan. These data demonstrate that the addition of direct viral oncolysis to the HSV-tk/GCV suicide gene system resulted in a striking improvement in treatment efficacy and that it may offer advantages over the use of chemotherapeutic agents for treatment of localized disease.