Replication-competent oncolytic viruses are being developed for human cancer therapy. We previously reported that an attenuated adenovirus (OBP-301, 'Telomelysin'), in which the hTERT promoter element drives expression of E1A and E1B genes linked with an IRES, could replicate in cancer cells, and causes selective lysis of cancer cells. We further constructed OBP-405 ('Telomelysin-RGD') that contains an RGD motif in the HI loop of the fiber knob. We examined whether OBP-405 could be effective in overcoming the limitations of OBP-301, specifically their inefficient infection into cells lacking the primary receptor, the coxsackievirus and adenovirus receptor (CAR). By flow cytometric analysis, H1299 (lung) and SW620 (colorectal) tumor cells showed high levels of CAR expression, whereas LN444 (glioblastoma), LNZ308 (glioblastoma), and H1299-R5 (lung) tumor cells were negative for CAR expression. A quantitative real-time PCR analysis demonstrated that fiber-modified OBP-405 infected more efficiently than OBP-301, although the intracellular replication rate of both viruses was consistent. The comparative antitumor effect of fiber-modified OBP-405 and unmodified OBP-301 for human cancer cells was evaluated in vitro by XTT assay as well as in vivo by using athymic mice carrying xenografts. OBP-405 had a profound oncolytic effect on human cancer cell lines compared to OBP-301, in particular on cells with low CAR expression. Intratumoral injection of 10(7) plaque-forming units of OBP-405 into CAR-negative H1299-R5 lung tumor xenografts in nu/nu mice resulted in a significant inhibition of tumor growth and long-term survival in all treated mice. Moreover, selective replication of OBP-405 in the distant, uninjected H1299-R5 tumors was demonstrated. Our results suggest that fiber-modified replication-competent adenovirus OBP-405 exhibits a broad target range by increasing infection efficiency, an outcome that has important implications for the treatment of human cancers.