Targeting death receptors with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has the remarkable potential to selectively kill malignant cells whereas normal cells are largely unaffected by this treatment. However, some tumor cells, including leukemia cells, exhibit resistance to this molecule. To investigate the basis for resistance of leukemia cells to the zinc-bound form of Apo2 ligand (Apo2L)/TRAIL, which is currently being evaluated in clinical trial, we isolated several resistant HL60 clones from parental HL60 cells by selection using the recombinant Apo2L/TRAIL. Differing resistance mechanisms were identified and characterized in these Apo2L/TRAIL-resistant clones. In one case, the level of the cell-surface death receptor DR4, but not DR5, was significantly decreased. However, these cells did undergo apoptosis in response to another form of recombinant TRAIL, histidine-tagged TRAIL, suggesting differing contributions of DR4 and DR5 in the response to these two forms of TRAIL. In the case of other clones, expression of procaspase-8 protein was lost and this was associated with a novel Leu(22)-->Phe(22) point mutation in CASP-8 gene. These results show that cells within a given tumor can have widely distinct mechanisms underlying resistance to Apo2L/TRAIL.