Replication-competent adenoviruses may provide a highly efficient means of delivering therapeutic genes to tumors. Previously, we evaluated in vitro a replication-competent adenovirus (Ad5-CD/TK rep) containing a cytosine deaminase (CD)/herpes simplex type 1 thymidine kinase (HSV-1 TK) fusion gene that allows lytic viral therapy to be combined with double suicide gene therapy. Both the CD/5-FC and HSV-1 TK/GCV enzyme/prodrug systems enhanced the tumor cell-specific cytopathic effects of the Ad5-CD/TK rep virus in vitro and sensitized cells to radiation. To extend these in vitro findings in vivo, we evaluated the antitumor activity of the Ad5-CD/TK rep virus in combination with double prodrug therapy and radiation therapy. The Ad5-CD/TK rep virus independently demonstrated significant antitumor activity against C33A cervical carcinoma xenografts. Therapeutic outcome was dramatically improved with systemic administration of double, but not single, prodrug (5-FC+GCV) therapy. When used in a neoadjuvant setting, Ad5-CD/TK rep-mediated double suicide gene therapy dramatically potentiated the effectiveness of radiation therapy. The trimodal approach of Ad5-CD/TK rep viral, double suicide gene, and radiotherapies produced significant tumor regression and ultimately 100% tumor cure. The results demonstrate the high therapeutic potential of the trimodal approach and provide a solid foundation for future clinical trials.