Miniglucagon (MG), the C-terminal glucagon fragment, processed from glucagon by the Miniglucagon-Generating Endopeptidase (MGE) at the Arg₁₇-Arg₁₈ dibasic site, displays biological effects opposite to that of the mother-hormone. This secondary processing occurs in the glucagon- and MG-producing -cells of the islets of Langerhans and from circulating glucagon. We first characterized the enzymatic activities of MGE in culture media from glucagon and MG-secreting TC1.6 cells as made of a metallo-endoprotease and an aminopeptidase. We observed that glucagon is a substrate for N-arginine dibasic convertase (NRDc), a metallo-endoprotease, and that Aminopeptidase B cleaves in vitro the intermediate cleavage products sequentially, releasing mature MG. Furthermore, immuno-depletion of either enzyme resulted in the disappearance of the majority of MGE activity from the culture medium. We found RNAs and proteins corresponding to both enzymes in different cell lines containing a MGE activity (mouse TC1.6 cells, rat hepatic FaO and rat pituitary GH₄C₁). Using confocal microscopy, we observed a granular immunostaining of both enzymes in the TC1.6 and native rat -cells from islets of Langerhans. By immunogold electron microscopy, both enzymes were found in the mature secretory granules of -cells, close to their substrate (glucagon) and their product (MG). Finally, we found NRDc only in the fractions from perfused pancreas that contain glucagon and miniglucagon after stimulation by hypoglycemia. We conclude that MGE is composed of NRDc and Aminopeptidase B acting sequentially, providing a molecular basis for this uncommon regulatory process, which should be now addressed in both physiological and pathophysiological situations.