Endothelial cells impart thromboresistance to the blood vessel wall. As modulators of fibrinolytic activity, these cells synthesize and secrete tissue plasminogen activator (t-PA) as well as its physiologic inhibitor, plasminogen activator inhibitor-1. In addition, endothelial cells support membrane-associated assembly of plasminogen and tissue plasminogen activator. Recently, an M(r) approximately 40,000 protein expressed on endothelial cells has been shown to interact noncompetitively through disparate mechanisms with both t-PA and plasminogen, suggesting trimolecular assembly of enzyme, substrate, and receptor (Hajjar, K. A. 1991. J. Biol. Chem. 266:21962-21970). In the present study, treatment of cultured endothelial cells with DL-homocysteine was specifically associated with a selective reduction in cellular binding sites for t-PA. This 65% decrease in binding was associated with a 60% decrease in cell-associated t-PA activity. No change in affinity for t-PA or plasminogen or in the maximal number of binding sites for plasminogen was observed. Matrix-associated t-PA binding sites were not affected. These data suggest a new mechanism whereby homocysteine may perturb endothelial cell function, thus promoting a prothrombotic state at the surface of the blood vessel wall.