The fibrinolytic activity of blood is regulated by expressing tissue-type plasminogen activator (t-PA) and its specific inhibitor, type-1 plasminogen activator inhibitor (PAI-1), from vascular endothelial cells. Since t-PA is a major plasminogen activator in blood, it is considered that the binding protein for t-PA, which exists on endothelial cell membrane, immobilizes t-PA on the surface of endothelial cells and enhances their antithrombotic property. Recently, we have found a new t-PA binding protein in endothelial cells. Its amino acid sequence has matched that of human adenine nucleotide translocase-1 (ANT1). The aims of this study are to confirm the binding of t-PA to ANT1, and to clarify the effect of ANT1 on fibrinolytic activity around endothelial cells. ANT1 is prepared from recombinant glutathione S-transferase (GST)-ANT1 fusion protein, and reveals t-PA binding activity in a ligand blot assay. In addition, ANT1 is exclusively expressed on endothelial cell membrane by using pDisplay vector. Interaction of t-PA with ANT1, which is expressed on the surface of endothelial cells, is confirmed by IAsys binding analysis and chromogenic assay. The heterologous expression of ANT1 on endothelial cell membrane enhances the t-PA binding ability of endothelial cells and the effect of ANT1 expression on fibrinolytic activity is demonstrated by increasing t-PA-catalyzed plasminogen activation. These results suggest that a novel t-PA-binding protein, ANT1, may concentrate t-PA on the surface of cells and enhance fibrinolytic properties around endothelial cells; therefore, ANT1 can be a powerful tool for regulating the plasminogen activation system in the vessel.