Cultured bovine aortic endothelial cells are associated with an unusually stable fibrinolytic inhibitor (Loskutoff, D.J., van Mourik, J.A., Erickson, L.A., and Lawrence, D. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2956-2960). This inhibitor was purified to apparent homogeneity from medium conditioned by these cells by a combination of concanavalin A affinity chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a single-chain glycoprotein of apparent Mr 50,000 +/- 2,500 and isoelectric point of 4.5-5.0, and inhibits the ability of both urokinase and tissue-type plasminogen activator to cleave and active plasminogen. This inhibition of plasminogen activator activity is associated with the formation of an enzyme-inhibitor complex which can be detected after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified inhibitor retains full activity after incubation in the presence of 0.1% sodium dodecyl sulfate, or at pH 2.7, two treatments which rapidly destroy the activity of protease nexin, another cellular inhibitor of fibrinolysis. The inhibitor purified from cloned endothelial cells cultured in the presence of L-[3,4,5-3H]leucine represented 2.5-12% of the total radiolabeled protein released by the cells in a 24-h period. These results indicate that cultured bovine aortic endothelial cells synthesize and secrete a protein which inhibits plasminogen activators and is distinct from protease nexin. It is a major endothelial cell product, and, as such, probably plays an important role in regulating the fibrinolytic system of these cells.