Recombinant adenoviral vectors (AdVTNF-alpha) expressing alpha tumor necrosis factor (TNF-alpha) under control of cytomegalovirus (CMV) promoter have been used in cancer gene therapy. To reduce its cytotoxicity, we constructed a recombinant AdV(TERT)mTNF-alpha expressing a mutant TNF-alpha (mTNF-alpha) with mutations at D142N and A144R under control of human telomerase reverse transcriptase (hTERT) promoter for treatment of well-established ovalbumin (OVA)-expressing murine B16 melanoma (BL6-10(OVA)) (6 mm in diameter). We demonstrated that the mTNF-alpha with mutations at D142N and A144R has less in vitro cytotoxicity, but maintains its functional effect in the stimulation of T-cell proliferation. The in vitro and in vivo transgene expressions under control of hTERT promoter are highly restricted in tumor cells compared with those under the control of the CMV promoter. AdV(TERT)mTNF-alpha gene therapy by intratumoral injection of AdV(TERT)mTNF-alpha vector (2 x 10(9) PFU) expressing the mutant mTNF-alpha under control of hTERT promoter reduces its in vivo toxicity, eradicates well-established BL6-10(OVA) tumors in 4/10 tumor-bearing mice, and induces OVA-specific CD8(+) T-cell-mediated long-term antitumor immunity. Therefore, AdV(TERT)mTNF-alpha gene therapy may be very useful in the immunotherapy of cancer.