Human neutrophil peptides (HNPs) are 29 to 30 amino acid antimicrobial peptides localized in the azulophil granules in neutrophils. We isolated endogenous molecular forms of HNPs from normal human bone marrow, plasma and peripheral blood neutrophils and determined their primary structures to investigate their post-translational processing. HNPs initially are synthesized as 94 amino acid precursors that produce 75 amino acid pro-HNPs by cleavage of a signal peptide. The majority of pro-HNPs was processed to 56 amino acid intermediates by preaspartate proteolytic cleavage in the bone marrow then to mature HNPs in peripheral blood neutrophils. Part of pro-HNPs was released into the plasma, in which they constituted 25-30% of the HNP molecules. Pro-HNPs and their mRNAs were detected both in peripheral blood neutrophils and bone marrow. Identification of the post-translational processing products of HNPs should provide a better understanding of the biosynthesis of the peptides.