Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is cytotoxic to the majority of cancer cells while sparing most normal cells. However, different prostate carcinoma cell lines respond with different sensitivities to TRAIL, urging us to disclose the mechanisms that determine TRAIL sensitivity in prostate cancer cells, i.e. to identify and validate target molecules. In this report, we show that down-regulation ('knockdown') of Bcl-xL, but not Bcl-2, markedly amplifies TRAIL-induced apoptosis in PC-3 prostate carcinoma cells. The knockdown was accomplished by second-generation chimeric antisense oligonucleotides: Bcl-2 and Bcl-xL levels were strongly and reproducibly reduced, as revealed by real-time RT-PCR and Western blot analyses. Knockdown of Bcl-xL and administration of TRAIL significantly synergized in dissipation of mitochondrial membrane potential, release of cytochrome c, activation of caspase-9 and -3 and, consequently, apoptotic cell death. Knockdown of Bcl-2 did not affect any of these activities. We conclude that that Bcl-xL represents a promising target to improve cancer therapy by potentiating TRAIL's cytotoxic effects.