Tumor necrosis factor-α (TNF-α), one of the major inflammatory cytokines, is known to influence endothelial cell migration. In this study, we demonstrate that exposure of calf pulmonary artery endothelial cells to TNF-α caused an increase in the formation of membrane protrusions and cell migration. Fluorescence microscopy revealed an increase in αvβ3focal contacts but a decrease in α5β1 focal contacts in TNF-α-treated cells. In addition, both cell-surface and total cellular expression of αvβ3-integrins increased significantly, whereas the expression of α5β1-integrins was unaltered. Only focal contacts containing αvβ3- but not α5β1-integrins were present in membrane protrusions of cells at the migration front. In contrast, robust focal contacts containing α5β1-integrins were present in cells behind the migration front. A blocking antibody to αvβ3, but not a blocking antibody to α5-integrins, significantly inhibited TNF-α-induced cell migration. These results indicate that in response to TNF-α, endothelial cells may increase the activation and ligation of αvβ3 while decreasing the activation and ligation of α5β1-integrins to facilitate cell migration, a process essential for vascular wound healing and angiogenesis.