Although adhesive interactions between metastasizing cancer cells and vascular endothelial cells are critical in hematogenous metastasis, the early molecular events of the cancer-endothelial interaction remain largely obscure. Here we investigated the functional impact of cancer cells on endothelial permeability. We examined the binding of human pancreatic carcinoma cells MIA PaCa-2, PANC-1 and PSN-1 to a human umbilical vein endothelial cell (HUVEC) monolayer and the subsequent changes in the transendothelial electronic resistance (TEER) of the HUVEC. We found that MIA PaCa-2 and PANC-1 cells preferentially bound to the tri-cellular corners of HUVEC and induced a rapid and irreversible reduction of TEER. The reduction of HUVEC TEER was associated with the focal disengagement of endothelial junctional adhesion molecules VE-cadherin and CD31. Blocking antibodies to integrin beta1, CD44, or CD9 affected neither the MIA PaCa-2 binding to HUVEC nor the reduction of TEER. Specific inhibitors for metalloproteinases, tyrosine-kinases and lipoxigenases, and a neutralizing anti-vascular endothelial growth factor antibody failed to affect the MIA PaCa-2-induced reduction of HUVEC TEER, whereas treatment of the cells with paraformaldehyde or cytochalasin B abrogated the TEER reduction. These findings indicate that the MIA PaCa-2 cells bind selectively to endothelial tri-cellular corners, triggering a reduction of HUVEC TEER, which requires the active metabolism and intact actin cytoskeleton of the carcinoma cells, and is apparently unrelated to previously described cell adhesion and soluble factor pathways. Our data indicate a novel cell-contact-dependent mechanism for the cancer cell-mediated breakdown of endothelial barrier functions, which may be important in hematogenous cancer metastasis.