Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family and a potent inducer of apoptosis. TRAIL has been shown to effectively limit tumor growth in vivo without detectable cytotoxic side-effects. Interferon (IFN)-γ often modulates the anticancer activities of TNF family members including TRAIL. However, little is known about the mechanism. To explore the mechanism, A549, HeLa, LNCaP, Hep3B and HepG2 cells were pretreated with IFN-γ, and then exposed to TRAIL. IFN-γ pretreatment augmented TRAIL-induced apoptosis in all these cell lines. A549 cells were selected and further characterized for IFN-γ action in TRAIL-induced apoptosis. Western blotting analyses revealed that IFN-γ dramatically increased the protein levels of interferon regulatory factor (IRF)-1, but not TRAIL receptors (DR4 and DR5) and pro-apoptotic (FADD and Bax) and anti-apoptotic factors (Bcl-2, Bel-XL, cIAP-1, cIAP-2 and XIAP). To elucidate the functional role of IRF-1 in IFN-γ-enhanced TRAIL-induced apoptosis, IRP-1 was first overexpressed by using an adenoviral vector AdIRF-1. IRF-1 overexpression minimally increased apoptotic cell death, but significantly enhanced apoptotic cell death induced by TRAIL when infected cells were treated with TRAIL. In further experiments using an antisense oligonucleotide, a specific repression of IRF-1 expression abolished enhancer activity of IFN-γ for TRAIL-induced apoptosis. Therefore, our data indicate that IFN-γ enhances TRAIL-induced apoptosis through IRF-1.