An E1B 55-kDa gene-defective adenovirus (Adv), ONYX-015, has been reported to be a highly useful replication-competent Adv that shows cytopathic effect for cancers with an abnormal p53 gene, without damaging normal tissues. In this study, we combined this Adv (Adv-E1AdB) with a fiber mutation, F/K20, which has a stretch of 20 lysine residues added at the COOH-terminus of the fiber and shows high transduction efficiency to gliomas. In U-373 MG glioma cells, the transduction efficiency of Adv-F/K20 for lacZ was nine times higher than that of the Adv with wild-type fiber (Adv-F/wt) for lacZ. At a multiplicity of infection of 30, the replication efficiency of Adv-E1AdB-F/K20 was 11 times higher than that of Adv-E1AdB with wt fiber (Adv-E1AdB-F/wt). The ED₅₀ value of Adv-E1AdB-F/K20 to U-373 MG cells, which is a measure of the in vitro cytopathic effect, was 32 times greater than that of Adv-E1AdB-F/wt. Injection of Adv-E1AdB-F/K20 suppressed the in vivo growth of tumors. The antitumoral effect of Adv-E1AdB-F/K20 was remarkably stronger than that of Adv-E1AdB-F/wt. A greater quantity of replicated virus protein (hexon) by infection with Adv-E1AdB-F/K20 was demonstrated in vitro and in vivo, compared with that of Adv-E1AdB-F/wt. In conclusion, gene therapy using Adv-E1AdB-F/K20, which drastically augmented the antitumoral effect of Adv-E1AdB, will be a promising therapeutic approach for gliomas.