Protease nexin-1 is a protein proteinase inhibitor that is secreted by a variety of cultured cells and rapidly forms complexes with thrombin, urokinase, and plasmin; the complexes then bind back to the cells and are internalized and degraded. In fibroblast cultures, protease nexin-1 is localized to the extracellular matrix. Here we report that protease nexin-1, which is bound to the surface of fibroblasts, forms complexes with thrombin, but not urokinase or plasmin. Experiments were conducted to determine directly if protease nexin-1 binding to the fibroblast surface alters its proteinase specificity. To do this, cell surface protease nexin-1 was inhibited using anti-protease nexin-1 monoclonal antibodies that stoichiometrically block its ability to form complexes with target proteinases. Then, purified protease nexin-1 was added to these cells; the cell-bound molecule formed complexes with thrombin, but not urokinase or plasmin. Similar experiments showed that protease nexin-1 bound to preparations of fibroblast extracellular matrix also formed complexes with thrombin, but not urokinase or plasmin. Components of the extracellular matrix other than heparin-like glycosaminoglycans are required for this regulation since heparin did not block the formation of complexes between protease nexin-1 and urokinase or plasmin. These results suggest that protease nexin-1 is primarily a thrombin inhibitor in interstitial fluids where much of it would be bound to cell surfaces.