Proapoptotic gene transfer to promote death or to augment killing by DNA-damaging agents represents a promising strategy for cancer therapy. We have constructed an adenoviral Tet-Off trade mark vector with tightly controlled expression of Bid (Ad-Bid) (Clontech, Palo Alto, CA). Using the non-small cell lung cancer cell lines H460, H358, and A549, low dose Ad-Bid was shown to induce high levels of full-length Bid as well as caspase-3 and -9 activity. Although only a small fraction of Bid was processed to truncated Bid (a step inhibited by benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone), Ad-Bid gene transfer resulted in mitochondrial changes consistent with apoptosis (mitochondrial depolarization, cytochrome c release), DNA fragmentation, and a dramatic loss of cell viability. The proapoptotic effects of Ad-Bid were independent of p53 status and were augmented markedly by caspase-8 activators such as the DNA-damaging agent cisplatin. When Ad-Bid and cisplatin were used together, chemosensitivity was restored in p53-null H358 cells, increasing death from 35% following treatment with cisplatin and Ad-LacZ to >90% death with Ad-Bid and cisplatin (Ad-Bid alone induced 50% cell death under these conditions). Ad-Bid can induce apoptosis in malignant cells and enhance chemosensitivity in the absence of p53, suggesting this approach as a potential cancer therapy.