Human monocyte/neutrophil elastase inhibitor (HEI) is a serpin superfamily protein that rapidly and irreversibly inhibits neutrophil and pancreatic elastase. We generated a recombinant baculovirus that supports production of HEI in insect cells at approximately 400 times the level in monocytes. Recombinant HEI was found to be indistinguishable from monocyte HEI in its physicochemical properties and ability to inhibit neutrophil elastase and pancreatic elastase. The recombinant protein was used to test for additional functions. HEI was shown to inhibit proteinase 3, an important neutrophil inflammatory protease, by the classical serpin mechanism of forming a covalent protease-protease inhibitor complex. Preliminary evidence suggests that HEI also inhibits neutrophil cathepsin G. On the other hand, HEI does not inhibit u-plasminogen activator (urokinase). These findings suggest that HEI functions as a class-specific regulator of the neutrophil serine proteases characteristically found at inflammatory sites.