The azurophil granules of neutrophil granulocytes contain neutral proteases such as leukocyte elastase and cathepsin G. These are synthesized as inactive precursors, but following proteolytic processing, they are stored in granules as active enzymes. We describe the establishment of a transgenic cellular model for expression of the human myeloid serine protease cathepsin G. The cDNA for preprocathepsin G was stably expressed in the rat basophilic/mast cell line RBL-1 and the translation product was characterized by use of biosynthetic labeling followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography. Conversion into complex form of an asparagine-linked carbohydrate unit of approximately 3.5 kDa was shown, as judged by the products obtained upon treatment with endoglycosidase H and N-glycanase. Proteolytic processing of 32.5-kDa procathepsin G into a 31-kDa form, within 1-2 h after synthesis, was demonstrated by pulse-chase experiments. Further processing into a 30-kDa form also occurred to a minor extent. The processed forms were enzymatically active, as judged by affinity for the serine protease inhibitors diisopropylfluorophosphate and aprotinin. Translocation of processed forms of cathepsin G to high density fractions, indicating targeting of the protease to granules, was demonstrated by subcellular fractionation. The weak base NH4Cl was shown to delay the processing and enzymatic activation of cathepsin G, whereas the monovalent ionophore monensin completely inhibited both events. Our data demonstrate that human cathepsin G transfected to rat RBL-1 cells, is proteolytically processed into enzymatically active forms and that subcellular transfer to granular organelles occurs. As the processing of transgenic human cathepsin G corresponds to that of endogenous protease of myeloid cells, the model should provide new unique possibilities to further characterize the activation and granular targeting of myeloid serine proteases.