Previously, we showed that the clotting factor thrombin binds to bovine corneal endothelial cells forming a covalent complex with a protein released by these cells into the culture media. We report that the plasminogen activator, urokinase, an enzyme involved in dissolution of blood clots, binds specifically to bovine corneal endothelial cells. [125I]-urokinase is rapidly bound by corneal endothelial cells. The serine active site of urokinase is required for binding since [125I]-urokinase inactivated with diisoprophylfluorophosphate does not bind to the cells. Pre-incubation of corneal cells with unlabeled urokinase for 20 hr results in increased release of binding sites for [125I]-urokinase into the culture media and at least a 3.5-fold increase in binding by the cells. [125I]-urokinase forms a 73 300 dalton sodium dodecyl sulfate complex with a corneal endothelial cell protein. Although thrombin competes with urokinase for binding to corneal endothelial cells, urokinase has at least a 10-fold higher affinity for binding to the cells. Furthermore, these cells make a plasminogen activator identical in molecular weight to urokinase. Thus, under physiological conditions, urokinase rather than thrombin binds to corneal endothelial cells. Binding may serve to regulate endogenous urokinase activity in the anterior chamber of the eye by limiting its extracellular proteolytic activity.