Two obstacles limiting the efficacy of nearly all cancer gene therapy trials are low gene transduction efficiencies and the lack of tumor specificity. Recently, a replication-competent, E1B-attenuated adenovirus (ONYX-015) was developed that could overcome these limitations, because it was capable of efficiently and selectively destroying tumor cells lacking functional p53. In an attempt to improve both the efficacy and safety of this approach, we constructed a similar adenovirus (FGR) containing a cytosine deaminase (CD)/herpes simplex virus type-1 thymidine kinase (HSV-1 TK) fusion gene, thereby allowing for the utilization of double-suicide gene therapy, which has previously been demonstrated to produce significant antitumor effects and potentiate the therapeutic effects of radiation. The FGR virus exhibited the same tumor cell specificity and replication kinetics as the ONYX-015 virus in vitro. Importantly, both the CD/5-FC and HSV-1 TK/GCV suicide gene systems markedly enhanced the tumor cell-specific cytopathic effect of the virus, and, as expected, sensitized tumor cells to radiation. By contrast, neither the FGR virus nor either suicide gene system showed significant toxicity to normal human cells. Both suicide gene systems could be used to suppress viral replication effectively, thereby providing a means to control viral spread. The results support the thesis that the three-pronged approach of viral therapy, suicide gene therapy, and radiotherapy may represent a powerful and safe means of selectively destroying tumor cells in vivo.