Tumor Necrosis Factor Receptor Apoptosis Inducing Ligand (TRAIL) appears as an interesting candidate for targeted cancer therapy as it induces apoptosis in cancer cells without toxicity to normal cells. TRAIL elicits apoptosis through agonist death receptor TRAIL-R1 and TRAIL-R2 engagement. Nevertheless, recombinant soluble TRAIL and monoclonal antibodies against these receptors demonstrated insufficient efficacy in clinical trials. This may be explained by the cell-type dependency of the apoptotic response, itself influenced by the effect on ligand binding mode of factors such as the level of receptor oligomerization or glycosylation. To investigate the relation between binding mode and signaling, we used previously described synthetic divalent and monovalent peptides specific for TRAIL-R2. We measured their pro-apoptotic activity on three cancer cell lines sensitive to rhTRAIL induced-apoptosis and monitored their cell-surface binding kinetics. The two divalent peptides bound with strong affinity to TRAIL-R2 expressed on B lymphoma BJAB cells and induced a high degree of apoptosis. By contrast, the same peptides bound weakly to TRAIL-R2 expressed at the surface of the human colon cancer HCT116 or T lymphoma Jurkat cell lines and did not induce their apoptosis. Cross-linking experiments suggest that these differences could be afforded by variations in the TRAIL-R2 oligomerization state at cell surface before ligand addition. Moreover divalent peptides showed a different efficiency in BJAB apoptosis induction, and kinetic distribution analysis of the BJAB binding curves suggested subtle differences in binding mechanisms. Thus our data support a relation between the cell-surface binding mode of the peptides and their pro-apoptotic activity. In this case the precise characterization of ligand binding to the surface of living cells would be predictive of the therapeutic potential of TRAIL-R2 synthetic ligands prior to clinical trials.