Vascular endothelium forms a dynamic interface between blood and underlying tissues. Endothelial monolayer integrity is required for controlled vascular permeability and to preclude exposure of subendothelial cell matrix to circulating cells. Recent studies have established that cultured human umbilical vein endothelial cells (ECs) express receptors for plasminogen (plg) and urokinase-like plasminogen activator (uPA). In the present study, we provide evidence that in EC, uPA receptor is present in focal contacts and at cell-cell contact sites. In these cells, addition of plg and uPA to confluent EC generates a retraction of the monolayer that is evidenced by loss of cell-cell contacts and increase in monolayer permeability. The phenomenon is reversible even after 6 hours of plg-uPA treatment. Inhibition of plg-uPA effect is obtained with plasmin inhibitors, as well as reagents that block binding of uPA or plg to the cell surface. The retractive effect of plg-uPA is concomitant to surface activation of plasminogen and to the loss of cell-cell activation of plg can induce EC retraction, possibly by causing proteolysis at specific cell-cell contacts and cell-matrix sites. This process may be important in mediating the passage of metastatic tumor cells through an intact EC monolayer as well as in regulating contacts between circulating cells and endothelium.