CV787, a novel highly prostate-specific replication-competent adenovirus with improved efficacy, was constructed. CV787 contains the prostate-specific rat probasin promoter, driving the adenovirus type 5 (Ad5) E1A gene, and the human prostate-specific enhancer/promoter, driving the E1B gene. To improve efficacy, we constructed CV787 such that it also contains the entire Ad5 E3 region. CV787 replicates in prostate-specific antigen (PSA)⁺ cells as well as wild-type adenovirus, but in PSA⁻ cells, CV787 replicates 10⁴–10⁵ times less efficiently. CV787 destroys PSA⁺ prostate cancer cells 10,000 times more efficiently than PSA⁻ cells. Incorporation of the Ad5 E3 region significantly improves the target cell killing ability or efficacy of CV787. In nu/nu mice carrying s.c. LNCaP xenografts, a single i.v. tail vein injection of CV787 eliminates 300-mm³ tumors within 4 weeks. CV787 could be a powerful therapeutic for human metastatic prostate cancer.