Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a novel member of the tumor necrosis factor superfamily of cytokines that induces cell death by apoptosis. TRAIL has been shown to be effective in almost two-thirds of solid tumors tested thus far, but its effect on pancreatic cancer cells is unknown. We tested the effect of TRAIL on seven human pancreatic cancer cell lines (HPAF, Panc1, Miapaca2, Bxpc3, Panc89, SW979, and Aspc1) in vitro. Of these cell lines, all but Aspc1 showed a significant dose-dependent increase in apoptosis. The apoptotic rate, as detected by a terminal deoxynucleotidyl transferase-mediated nick end labeling assay, was highest in Bxpc3 (71.5%), followed by HPAF (38.0%), Miapaca2 (24.9%), Panc1 (16.1%), Panc89 (15.8%), SW979 (13.9%), and Aspc1 (5.2%). Multiple treatments were more effective than a single treatment and caused a sustained and profound cell death in all but Aspc1 cells. There was no correlation between the effect of TRAIL and the differentiation grade of the cell lines, p53 mutation, or bcl-2 or bax expression. The resistance of Aspc1 cells to TRAIL was not related to the lack of TRAIL receptors. The combination of actinomycin D and TRAIL induced an almost complete lysis of Aspc1 cells, whereas actinomycin D alone had no effect on cell survival but inhibited the expression of the Flice inhibitory protein, which is assumed to play a role in the apoptotic pathway of TRAIL. Thus, the combination of actinomycin D and TRAIL appears to be a promising approach for the therapy of pancreatic cancers resistant to TRAIL.