Using a binary adenoviral system, we recently showed that the human telomerase reverse transcriptase (hTERT) promoter induces tumor-specific Bax gene expression. However, the strong cytotoxicity of Bax and other pro-apoptotic genes to packaging 293 cells has so far hindered construction of the desired single adenoviral vectors expressing toxic genes. We report here the construction of a single bicistronic adenoviral vector for tumor-specific Bax expression. The vector (Ad/gBax) utilizes the Tet-Off system and expresses a GFP/Bax fusion protein for easy detection. The hTERT promoter drives the expression of tTA, a transactivator capable of binding to TRE (tetracycline-responsive element) in the absence of tetracycline, which in turn induces expression of the GFP-Bax gene. The addition of tetracycline in 293 cells blocks the binding of tTA to TRE and substantially inhibits GFP-Bax expression and toxicity, thus allowing the packaging and production of Ad/gBax. Our data show that Ad/gBax could drive the high expression of GFP-Bax in tumor cells but not in normal cells and mouse tissues. Furthermore, the expression of GFP-Bax fusion protein elicited tumor-specific apoptosis in a variety of human cancer cells in vitro and in vivo at a level comparable to that induced by the binary system. Thus, Ad/gBax may become a potent therapeutic agent for the treatment of cancers.