Gene therapy is hampered by poor gene transfer to the tumor mass. We previously proposed a combination adenoviral gene therapy containing a conditionally replicating adenovirus (CRAD) expressing mutant E1 (Δ24RGD) and a replication-defective E1-deleted adenovirus to enhance the efficiency of gene transfer. Mutant E1 expressed by Δ24RGD enables the replication of replication-defective adenoviruses in tumors when cancer cells are co-infected with both viruses. In this study, gene transfer rates in xenografts tumors were monitored by bioluminescence in cells infected with the replication-defective adenovirus-luciferase (ad-luc). Tumor masses treated with CRAD + ad-luc showed dramatically stronger and more prolonged luciferase expression than ad-luc-treated tumors and this expression spread through the entire tumor mass without significant systemic spread. Transduction with CRAD + replication-defective adenovirus-p27 increased the expression of p27 by 24-fold versus transduction with ad-p27 alone. Treatment of a lung cancer cell line and of established lung cancer xenografts with CRAD + adenovirus-p27 also induced stronger growth suppression than treatment with either virus alone. These findings confirm the selective replication of E1-deleted adenovirus containing a therapeutic gene due to the presence of mutant E1 produced by Δ24RGD in tumors. Moreover, this replication increased the therapeutic gene transfer rate and enhanced its antitumor effects.