Vascular endothelial cells regulate the fibrinolytic system in blood by expressing cell-surface receptors for plasminogen and plasminogen activators (PAs) as well as secreting PAs and their inhibitors. Although several receptors for plasminogen and PAs have been identified in many cell types, little is known about tissue-type PA (t-PA)-specific receptor (t-PAR) on endothelial cells except a few reports. By using suspended human umbilical vein endothelial cells (HUVEC), which are free from the formation of extracellular matrix (ECM)--where type-1 plasminogen activator inhibitor (PAI-1) preferably accumulates and interacts with t-PA with high affinity--we demonstrated a relatively low affinity binding site for t-PA on the cells and identified a novel t-PAR. The isolation and characterization of HUVEC-derived t-PAR was performed in the present study. A 20-kDa t-PAR was successively isolated and purified by high performance liquid chromatography system from HUVEC which specifically binds t-PA and not plasminogen forming a 90-kDa complex with t-PA. When t-PA binds the immobilized t-PAR stoichiometrically 1:1, the enzymatic activity of t-PA was enhanced 90-fold. Thus, it is suggested that the t-PAR may function as a t-PA-enhancing molecule expressed on the surface of endothelial cells.