To overcome the low efficiency of gene therapy, we combined a conditionally replicating adenovirus (CRAd) and an adenoviral vector with a therapeutic gene. CRAd has an oncolytic activity in cancer cells with abnormal Rb activity and helps the replication of therapeutic genes incorporated in the E1-deleted adenovirus. We investigated the anticancer effect of a combination of CRAd and adenovirus carrying tumor necrosis factor-related apoptosis inducing ligand (ad-TRAIL). We expected to see increased gene expression in cancer cells as well as an antitumor effect. With the combined application of CRAd and ad-luciferase in head and neck cancer cell lines, we observed considerably increased luciferase activity that was 10- to 50-fold greater than with ad-luciferase alone. The combination of CRAd and ad-TRAIL showed significant suppression of growth in cell lines and increased the sub-G1 portion of cells 30-fold compared to any single treatment. The expression of TRAIL was highly amplified by the combined treatment and was accompanied by expression of molecules related to apoptosis. In a xenograft animal model, mice treated with CRAd and ad-TRAIL showed complete regression of established tumors, whereas mice treated with CRAd or ad-TRAIL alone did not. In conclusion, this combined strategy using CRAd and adenovirus carrying a therapeutic gene increased the gene transfer rate and enhanced antitumor effects. We expect that this combination strategy could be extended to a multitarget cancer gene therapy by combining multiple adenoviruses and CRAd. (Cancer Sci 2009)