The host defense roles of neutrophil elastase in a porcine skin wound chamber model were explored. Analysis of wound fluid by acid-urea polyacrylamide gel electrophoresis, Western blot, and bacterial overlay confirmed that the neutrophil-derived protegrins constituted the major stable antimicrobial polypeptide in the wound fluid. The application to the wound of 0.10 and 0.25 mM N-methoxysuccinyl-alanine-alanine-proline-valine (AAPV) chloromethyl ketone, a specific neutrophil elastase inhibitor (NEI), blocked the proteolytic activation of protegrins and diminished the associated antimicrobial activity as detected by radial diffusion assay againstStaphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans or by bacterial gel overlay against S epidermidis and E coli. The application of the related cathepsin G inhibitor (CGI), benzyloxycarbonyl-glycine-leucine-phenylalanine (ZGLF) chloromethyl ketone, had no effect. In wound chambers that received 10⁶ colony-forming unit (CFU)/mL of S epidermidis, the presence of NEI significantly decreased the 24-hour clearance of bacteria from the wound compared to wounds treated with CGI or solvent only. Neither inhibitor, at 0.10 or 0.25 mM concentration, affected leukocyte accumulation or degranulation in the wound chambers. The in vitro microbicidal decrement due to NEI was restored by an amount of the specific protegrin (PG-1), which was equivalent to the measured difference of protegrin between control and inhibited chambers. Administration of 1 μg/mL exogenous PG-1 4 hours after chamber preparation was sufficient to normalize in vivo antimicrobial activity. Although pharmacologic NEIs are promising candidates as anti-inflammatory drugs, they may impair host defense in part by inhibiting the activation of cathelicidins by neutrophil elastase.