A protein of an estimated molecular mass (M,) of 18 000 has been identified as proglucagon in biosynthetic studies on isolated rat pancreatic islets [11. Moreover, a 10 000 M, peptide was recognized as a major conversion product of this prohormone. The intracellular accumulation of this radioactively labeled fragment, which was found to be devoid of the glucagon sequence, was accompanied by that of newly synthesized glucagon [11. Here, this major proglucagon fragment is identified as an abundant peptide in the Coomassie blue-stained electrophoretic patterns of rat islet proteins by the following criteria:(i) Its regional distribution in the rat pancreas corresponds to the difference in glucagon biosynthesis in ‘duodenal’and ‘splenic’islets, respectively;(ii) Its electrophoretic and isoelectric characteristics are identical to that of the major conversion product of [35S] methionine-labeled proglucagon. Moreover, secretion of the newly synthesized major fragment is found to be stimulated by arginine, its release following kinetics similar to that of labeled glucagon. These data strongly support the identification of the major proglucagon fragment as an endproduct, besides glucagon itself, of prohormonal proteolytic conversion.