To study the tumor specificity and antitumor activity of the replication-competent oncolytic adenovirus TOA02, which is controlled by a modified human telomerase reverse transcriptase (hTERT) promoter and expresses granulocyte macrophage colony-stimulating factor (GM-CSF). The wild-type hTERT promoter was modified, by inserting two E2F-binding sites. The effect of the modified hTERT on the viral yield and cytotoxicity of TOA02 were determined in vitro with a panel of tumor cells and normal cells, to evaluate tumor specificity; the effect on the antitumor efficacy and toxicity of TOA02 were determined in vivo, to evaluate the therapeutic potential of the adenovirus. The TOA02 adenovirus, which contained the modified hTERT promoter, produced a higher yield of virus in telomerase-positive and retinoblastoma-defective human cells, and a lower yield of virus in normal human cells than the wild-type adenovirus. A single injection of TOA02 showed strong antitumor efficacy in nude mice with human head/neck and hepatocellular carcinoma xenografts, and the efficacy further improved when used in combination with chemotherapy and with different routes of administration and regimens. In immunocompetent mice, the addition of GM-CSF produced a stronger antitumor activity and induced more mature dendritic cells and macrophages. The TOA02 adenovirus showed strong tumor-cell selectivity in vitro and antitumor efficacy in mouse models of human head/neck and hepatocellular cancer, suggesting that TOA02 has potential clinical applications for the treatment of solid tumors.