Tumor necrosis factor α (TNF-α) is a multifunctional cytokine with direct antitumor activity. However, clinical trials using TNF-α for cancer treatment have been disappointing due in part to its severe side effects, and it has been estimated that TNF-α therapy would be effective only at 5–25 times the maximum tolerated dose. We have recently modified a tetracycline (Tc) repressor/operator-based mammalian gene expression system and have generated a Tc-responsive recombinant adenovirus vector, AdVtTA.TNF-α. A variety of human tumor cells and T lymphocytes transduced by AdVtTA.TNF-α secreted high-titer (5,000–100,000 pg/10⁶ cells/24 h) and biologically active TNF-α in the absence of Tc. Expression of TNF-α in the transduced cells was nondetectable when the culture medium contained as little as 0.1 µg/ml of Tc. At least a fraction of the clonogenic cells from human peripheral blood stem cell concentrates were also transducible by AdVtTA.TNF-α. The availability of this type of adenovirus vector opens a door to tumor- or organ-specific delivery of high-dose TNF-α and other therapeutic gene products for systemic cancer gene therapy.