A potentially promising treatment of metastatic cancer is the systemic delivery of oncolytic adenoviruses. This requires engineering viruses which selectively replicate in tumors. We have constructed such an oncolytic adenovirus, OAS403, in which two early region genes are under the control of tumor-selective promoters that play a role in two key pathways involved in tumorigenesis. The early region E1A is controlled by the promoter for the E2F-1 gene, a transcription factor that primarily upregulates genes for cell growth. The E4 region is under control of the promoter for human telomerase reverse transcriptase, a gene upregulated in most cancer cells. OAS403 was evaluated in vitro on a panel of human cells and found to elicit tumor-selective cell killing. Also, OAS403 was less toxic in human hepatocyte cultures, as well as in vivo when compared to an oncolytic virus that lacked selective E4 control. A single intravenous injection of 3 x 10(12) vp/kg in a Hep3B xenograft mouse tumor model led to significant antitumor efficacy. Additionally, systemic administration in a pre-established LNCaP prostate tumor model resulted in over 80% complete tumor regressions at a tolerable dose. Vector genome copy number was measured in tumors and livers at various times following tail vein injection and showed a selective time-dependent increase in tumors but not livers over 29 days. Furthermore, efficacy was significantly improved when OAS403 treatment was combined with doxorubicin. This virus holds promise for the treatment of a broad range of human cancers including metastatic disease.