TNF-related apoptosis-inducing ligand (TRAIL, also known as Apo-2L) is a promising novel anticancer agent that selectively induces apoptosis in tumour cells and the activity of which can be enhanced by combined treatment with chemo- or radiotherapy. For therapeutic purposes, the use of full-length TRAIL may be favourable to recombinant TRAIL based on its increased tumour cell killing potential, and the delivery of TRAIL at the tumour site by adenovirus vectors may provide an approach to overcome the short half-life of recombinant TRAIL and hepatocyte toxicity in vivo. Here, we constructed an adenoviral vector expressing full-length TRAIL (AdTRAIL) and studied the potential of chemo- and radiotherapy in enhancing AdTRAIL-induced apoptosis in non-small cell lung cancer (NSCLC) H460 cells and normal cells and, in addition, investigated the mechanism of AdTRAIL-induced apoptosis. AdTRAIL effectively killed H460 cells, which we previously showed to have a deficiency in mitochondria-dependent apoptosis by downstream activation of caspase-8 rather than caspase-9. Further analyses revealed that AdTRAIL induces death receptor- and mitochondria-dependent apoptosis that could be partially suppressed by Bcl2 overexpression. Combined treatment with doxorubicin (DOX), cisplatin (CDDP), paclitaxel (PTX) and radiation strongly enhanced AdTRAIL-induced cytotoxicity in a synergistic way. Synergy was accompanied by the cleavage of Bid and an increase in caspase-8 processing that was abolished by Bcl2 overexpression, indicating that the Bid-mitochondrial amplification loop is functional in H460 cells. Moreover, combination treatment did not alter the tumour selectivity of AdTRAIL since normal human fibroblasts (NHFs) remained resistant under these conditions. These findings further indicate that the combined use of chemo/radiotherapy and adenovirus-produced full-length TRAIL may provide a valuable treatment option for NSCLC.