Neuroblastoma cells have been reported to be resistant to death induced by soluble, recombinant forms of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (CD253/TNFSF10) because of low or absent expression of caspase-8 and/or TRAIL-receptor 2 (TRAIL-R2/DR5/CD262/TNFRSF10b). However, their sensitivity to membrane-bound TRAIL on natural killer (NK) cells is not known. Comparing microarray gene expression and response to NK cell-mediated cytotoxicity, we observed a correlation between TRAIL-R2 expression and the sensitivity of 14 neuroblastoma cell lines to the cytotoxicity of NK cells activated with interleukin (IL)-2 plus IL-15. Even though most NK cytotoxicity was dependent upon perforin, the cytotoxicity was supplemented by TRAIL in 14 of 17 (82%) neuroblastoma cell lines as demonstrated using an anti-TRAIL neutralizing antibody. Similarly, a recently developed NK cell expansion system employing IL-2 plus lethally irradiated K562 feeder cells constitutively expressing membrane-bound IL-21 (K562 clone 9.mbIL21) resulted in activated NK cells derived from normal healthy donors and neuroblastoma patients that also utilized TRAIL to supplement cytotoxicity. Exogenous interferon-γ upregulated expression of caspase-8 in 3 of 4 neuroblastoma cell lines and increased the contribution of TRAIL to NK cytotoxicity against 2 of the 3 lines; however, relatively little inhibition of cytotoxicity was observed when activated NK cells were treated with an anti-interferon-γ neutralizing antibody. Constraining the binding of anti-TRAIL neutralizing antibody to membrane-bound TRAIL but not soluble TRAIL indicated that membrane-bound TRAIL alone was responsible for essentially all of the supplemental cytotoxicity. Together, these findings support a role for membrane-bound TRAIL in the cytotoxicity of NK cells against neuroblastoma cells.