Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent activator of the cell death pathway and exerts tumoricidal activity in vivo with minimal toxicity. In order to investigate the therapeutic potential of TRAIL in B chronic lymphocytic leukemia (B-CLL) we have analyzed the expression of TRAIL receptors (TRAIL-Rs) in leukemic cells from B-CLL patients and their in vitro sensitivity to apoptosis induced by recombinant human TRAIL. We have found TRAIL-R1 and -R2 death receptor, and TRAIL-R3 and -R4 decoy receptor mRNA expression in most of the 57 B-CLL patients studied (R1 82%, R2 100%, R3 96% and R4 82%). TRAIL-R1 and R2 proteins were expressed on the surface and within the cells, whereas R3 and R4 decoy receptors were almost exclusively expressed in the cytoplasm. Despite TRAIL death receptor expression, B-CLL cells were relatively resistant to induction of apoptosis by recombinant human TRAIL (300 ng/ml). However, the susceptibility to TRAIL-induced apoptosis was increased by treatment of B-CLL cells with actinomycin D (Act D). Western blot analysis showed higher constitutive expression of the long form of FLICE-inhibitory protein (FLIP(L)) in B-CLL as compared to normal tonsillar B cells. Act D treatment down-regulated both long and short FLIP expression, which was correlated with the increase in B-CLL sensitivity to TRAIL. Although the surface TRAIL death receptor expression was up-regulated both by cell culture and by Act D treatment, the changes were not correlated with a gain in susceptibility to TRAIL. In addition, neither decoy receptors nor Bcl-2 expression were affected by Act D. Our findings suggest the possible involvement of FLIP in regulating TRAIL-mediated apoptosis in B-CLL.