Neutrophils contain an assembly of granules destined for regulated secretion, each granule type with distinct constituents formed before terminal differentiation. The earliest granules are designated azurophil (primary), followed in time by specific (secondary), and gelatinase granules as well as secretory vesicles. Transcription factors regulate the genes for the granule proteins to ensure that expression of the gene products to be stored in different organelles is separated in time. Similar to lysosomal enzymes, many granule proteins, in particular those of the heterogeneous azurophil granules, are trimmed by proteolytic processing into mature proteins. Rodent myeloid cell lines have been utilized for research on the processing and targeting of human granule proteins after transfection of cDNA. Results from extensive work on the hematopoietic serine proteases of azurophil granules, employing in vitro mutagenesis, indicate that both an immature and a mature conformation are compatible with targeting for storage in granules. On the other hand, the amino-terminal propeptide of myeloperoxidase facilitates both the export from the endoplasmic reticulum and targeting for storage in granules. Similarly, targeting of defensins rely on an intact propeptide. The proteolytic processing into mature granule protein is most commonly a post-sorting event. Mis-sorting of specific granule proteins into azurophil or lysosome-like granules can result in premature activation and degradation, but represents a potential for manipulating the composition and function of neutrophil granules.