To explore the molecular mechanisms by which glioblastomas are resistant to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), we examined TRAIL signalling pathways in the tumours. TRAIL has four membrane-anchored receptors, death receptor 4/5 (DR4/5) and decoy receptor 1/2 (DcR1/2). Of these receptors, only DR5 was expressed consistently in glioblastoma cell lines and tumour tissues, ruling out the role of DcR1/2 in TRAIL resistance. Upon TRAIL binding, DR5 was homotrimerized and recruited Fas-associated death domain (FADD) and caspase-8 for the assembly of death-inducing signalling complex (DISC) in the lipid rafts of the plasma membrane. In the DISC, caspase-8 was cleaved and initiated apoptosis by cleaving downstream caspases in TRAIL-sensitive glioblastoma cells. In TRAIL-resistant cells, however, DR5-mediated DISC was modified by receptor-interacting protein (RIP), cellular FADD-like interleukin-1beta-converting enzyme inhibitory protein (c-FLIP) and phosphoprotein enriched in diabetes or in astrocyte-15 (PED/PEA-15). This DISC modification occurred in the non-raft fractions of the plasma membrane and resulted in the inhibition of caspase-8 cleavage and activation of nuclear factor-kappaB (NF-kappaB). Treatment of resistant cells with parthenolide, an inhibitor of inhibitor of kappaB (I-kappaB), eliminated TRAIL-induced NF-kappaB activity but not TRAIL resistance. In contrast, however, targeting of RIP, c-FLIP or PED/PEA-15 with small interfering RNA (siRNA) led to the redistribution of the DISC from non-rafts to lipid rafts and eliminated the inhibition of caspase-8 cleavage and thereby TRAIL resistance. Taken together, this study indicates that the DISC modification by RIP, c-FLIP and PED/PEA-15 is the most upstream event in TRAIL resistance in glioblastomas.