A major obstacle to the success of gene therapy strategies that directly target cancer cells is the poor vector distribution within solid tumors. To address this problem, we developed an E1b 55 kDa attenuated, replication-competent adenovirus (Ad.TKRC) which expresses the herpes simplex-1 thymidine kinase (HSVtk) gene to sensitize tumors to ganciclovir (GCV). Efficacy of this combined strategy was tested in nude mice with subcutaneous human A375 melanoma and ME180 cervical carcinomas. Intratumoral injection of a replication-defective adenoviral vector expressing HSVtk (Ad.TK) followed by GCV treatment resulted in doubling of the survival time of mice bearing A375 tumors and 20% long-term survival of mice with ME180 tumors. Treatment of tumors with Ad.TKRC without GCV resulted in a similar antitumor effect, confirming that the replicating vector has an oncolytic effect. When GCV was initiated 3 days after Ad.TKRC injection, survival of mice with each tumor type was greatly prolonged, with 60% of animals with ME180 tumors surviving for over 160 days. These results confirm that both the oncolysis caused by a replicating virus and suicide/prodrug gene therapy with HSVtk/GCV have potent antitumor effects. When combined, these two approaches are complementary resulting in a significantly improved treatment outcome.