Antimicrobial peptides of several structural classes have been found in phagocytes and epithelial cells of many animals. The broadly microbicidal protegrins (PG1, -2, and -3) were originally isolated as 16 to 18-amino-acid peptides from pig neutrophil lysates, but the corresponding cDNA sequences encoded much larger precursors that belonged to the cathelicidin family of antimicrobial peptides. We explored the storage, secretion, and microbicidal activation of protegrins in porcine neutrophils and in a model system consisting of recombinant proprotegrin 3 (pPG3) and various serine proteases and their inhibitors. Protegrins were stored in neutrophils as inactive proforms that were cleaved by neutrophil elastase to mature protegrins during the preparation of granule lysate and during phorbol myristate acetate-stimulated granule secretion from intact neutrophils. Recombinant pPG3 was efficiently cleaved by trace amounts of human neutrophil elastase or equivalent amounts of elastase activity from porcine neutrophils, but pPG3 was relatively resistant to porcine pancreatic elastase or human neutrophil cathepsin G. The recombinant pPG3 and neutrophil proprotegrins lacked microbicidal activity, but the mature protegrins generated in the elastase-mediated cleavage reaction were as active against Listeria monocytogenes as the chemically synthesized protegrin. The secretion and elastase-mediated activation of proprotegrins accounted for much of the stable microbicidal activity of porcine neutrophil secretions against L. monocytogenes. Secreted proprotegrins and trace amounts of elastase constitute a binary microbicidal system that is likely to contribute to the antimicrobial activity of porcine inflammatory fluids.