Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in cancer cells but not in normal cells. However, studies have indicated that more than half of human tumors exhibit TRAIL resistance. Although the mechanism of TRAIL resistance is not understood, it represents a barrier to any TRAIL-mediated gene therapy approach. In addition, no correlation between TRAIL receptor (TRAIL-R) expression profile and TRAIL resistance has been demonstrated in cancer cells. In this study, three different lung cancer cell lines and three different primary cell cultures established from patients with lung cancer (two patients with squamous cell lung carcinoma and one with adenocarcinoma) were screened for sensitivity to adenoviral delivery of TRAIL. Whereas TRAIL-resistant primary lung cell cultures and the A549 lung cancer cell line exhibited high levels of surface decoy receptor-2 (DcR2/TRAIL-R4) expression, TRAIL-sensitive lung cancer cell lines (HBE and H411) failed to express it. A DcR2 short interfering RNA (siRNA) approach involving three different siRNA constructs in combination downregulated DcR2/TRAIL-R4 expression and sensitized lung cancer cells to TRAIL-induced apoptosis. Immunohistochemical staining of samples from 10 patients with lung carcinoma suggested that high-level DcR2/TRAIL-R4 expression is a common phenotype observed in patients with non-small cell lung carcinoma.