Although Tumor necrosis factor-related apop- tosis-inducing ligand (TRAIL) selectively induces apoptosis of various cancer cells, some caner cell lines are resistant to TRAIL-induced cell death. To investigate the molecular mechanisms underlying TRAIL-resistance, two human breast cancer cell lines, MCF-7 (resistant to TRAIL) and MDA-MB-231 (sensitive to TRAIL), were used as a model system to analyze the different sensitivities to TRAIL cyto- toxicity. PKCδ inhibitor rottlerin, but not MEK and ERK1/2 inhibitor U0126 nor PI3K inhibitor LY294002, was shown to enhance TRAIL-induced apoptosis in MCF-7 cells signifi- cantly, suggesting that PKCδ might play an important role in the resistance of MCF-7 cells to TRAIL. In contrast, rottlerin, U0126, and Ly294002 had no effect on MDA-MB-231 apop- tosis induced by TRAIL under the same conditions. Further experiment showed that the combination of rottlerin and TRAIL cleaved PARP in the MCF-7 cells synergistically, but not in the MDA-MB-231 cells. The role of PKCδ in TRAIL-resistant MCF-7 cells was confirmed by knocking down the endogenous PKCδ expression using RNAi technol- ogy. Furthermore, caspase-3 reconstitution in MCF-7 cells was unable to alter PKCδ expression, suggesting that innate caspase-3 deficient in the cells does not cause PKCδ high expression. These data provide evidence for the first time that PKCδ plays a critical role in breast cancer cell lines to TRAIL cytotoxicity. To investigate the molecular mechanisms underlying TRAIL-induced cell death. To investigate the molecular mechanisms of TRAIL-resistance, two human breast cancer cell lines, MCF-7 (resistant to TRAIL) and MDA-MB-231 (sensitive to TRAIL), were used as a model system to analyze the different sensitivities to TRAIL cyto- toxicity. PKCδ inhibitor rottlerin, but not MEK and ERK1 / 2 inhibitor U0126 nor PI3K inhibitor LY294002, was shown to enhance TRAIL-induced apoptosis in MCF-7 cells signifi- cantly, suggesting that PKCδ might play an important role in the resistance of MCF-7 cells to TRAIL. In contrast, rottlerin, U0126 , and Ly294002 had no effect on MDA-MB-231 apop- tosis induced by TRAIL under the same conditions. Further experiment showed that the combination of rottlerin and TRAIL cleaved PARP in the MCF-7 cells synergistically, but not in the MDA-MB -231 cells . The role of PKCδ in TRAIL-resistant MCF-7 cells was confirmed by knockdown of the endogenous PKCδ expression using RNAi technology. Furthermore, caspase-3 reconstitution in MCF-7 cells was unable to alter PKC δ expression, suggesting that innate caspase -3 deficient in the cells does not cause PKCδ high expression. These data provide evidence for the first time that PKCδ plays a critical role in breast cancer cell lines to TRAIL cytotoxicity.