Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, is unique in its ability and mechanism of inhibiting serine proteases of a broad range of substrate specificity. However, although the catalytic domain of human urokinase-type plasminogen activator (uPA) has 40% identity to bovine trypsin and the substrate specificities of these two proteases are virtually identical, ecotin inhibits uPA almost 10,000-fold less efficiently than trypsin. Ecotin was expressed on the surface of filamentous bacteriophage (ecotin phage) to allow the isolation of more potent inhibitors of uPA from a library of ecotin variants. The 142-amino acid inhibitor was fused to the C-terminal domain of the M13 minor coat protein, pIII, through a Gly-Gly-Gly linker and assembled into phage particles. The ecotin phage were shown to react with anti-ecotin antibodies, revealing a stoichiometry of approximately one ecotin per bacteriophage. The ecotin displayed on the surface of phage inhibited trypsin with an equilibrium dissociation constant of 6.7 nM, in close approximation to that of free ecotin, indicating that phage-associated ecotin is correctly folded and functionally active. Reactive-site amino acids 84 and 85 of ecotin were then randomized and a library of 400 unique ecotin phage was created. Three hundred thousand members of the library were screened with immobilized uPA and subjected to three rounds of binding and in vitro selection. DNA sequence analysis of the selected ecotin phage showed that ecotin M84R/M85R predominated while ecotin M84R, M84K, and M84R/M85K were present at a lower frequency. The four ecotin variants were overexpressed and purified and their affinities toward uPA were determined. Each of the selected ecotin variants exhibited increased affinity for uPA when compared to wild-type ecotin with ecotin M84R/M85R showing a 2800-fold increase in binding affinity.