One of the mechanisms by which endothelial cells (ECs) regulate fibrinolysis is through the regulated assembly of proteins such as plasminogen, tissue plasminogen activator (tPA) and urokinase (uPA) on their membrane surface. Receptors for many of these fibrinolytic factors have been isolated and characterized. A unique 45 kD plasminogen receptor present on ECs derived from vein vasculature has been identified and resolved into two plasminogen binding components. One component consists of the unique 45 kD plasminogen receptor (pI = 6.3) whereas the other component (pI = 5.1) is identified as the cytoskeletal protein, actin. Immunofluorescent studies of isolated ECs confirm the presence of actin on their extracellular surface. This observation is consistent with a number of other recent reports of actin externally localized on other cell types. In vitro studies using purified actin confirm that plasminogen binds to actin both saturably and with relatively high affinity. Competition studies with lysine indicated that the binding was largely kringle-dependent, and when binding of tPA to actin was assessed, it also bound to actin with 70-80% of binding inhibited by lysine. Lipoprotein (a), which shows homology with plasminogen, also interacted with actin. Addition of plasminogen and low-density lipoproteins inhibited Lp(a) binding to actin in a dose-dependent fashion. Moreover, in competition with tPA, partial inhibition of plasminogen binding to actin was also observed. In experiments using anti-actin antibodies added in excess to cultured ECs, binding of plasminogen was inhibited by 45%, tPA binding was inhibited by 46% and Lp(a) binding was reduced by 56%, confirming actin as a binding site for these various ligands whilst attesting to the presence of other EC receptors for these proteins. Collectively, the data presented are consistent with actin playing a major role in localizing binding not only of plasminogen, but also of tissue plasminogen activator and Lp(a) to the surface of human endothelial cells.