Processing of the human enteric a-defensin, HD5 occurs extracellularly and is mediated by trypsin [30] , whereas mouse Paneth cell a-defensins are processed intracellularly by matrix metalloprotease 7 (MMP7, matrilysin) [31] . Neutrophil elastase (NE), an azurophil granule protease, was shown to correctly process RMAD4, a rhesus macaque neutrophil a-defensin in vitro [32] . Bovine and human cathelicidins, a distinct class of antimicrobial peptides, are processed by the azurophil granule proteases, NE [33] and proteinase 3 (PR3) [34] respectively. Studies with HL-60 cells, a human acute promyelocytic leukemia cell line, show that HNPs are biosynthesized and processed to mature peptides in these cells [35] , but the proteases involved in maturation of human myeloid a-defensins have not been identified. Because HNPs co-localize to neutrophil granules containing the serine proteases NE, PR3 and cathepsin G (CG) [36] , we hypothesized that HNP maturation is mediated by one or more of these proteinases. In this study, recombinant proHNP1 was used as substrate to analyze the human a-defensin convertase activities of NE, PR3, and CG. The requirement for the native HNP1 fold was analyzed by assessing the processing of folded and misfolded preparations of proHNP1. In addition, the antimicrobial activity of the resulting HNP-1 peptides was analyzed utilizing Staphylococcus aureus as the test organism.