Abstract To study the mechanism of degradation of glucagon with purified insulin-glucagon protease, glucagon was reacted with the enzyme at various times of incubation. The proteolysis was followed by the production of flourescamine-reacting material as well as reaction with dansyl chloride, cleavage by acid hydrolysis, and identification by thin layer chromatography. For quantitative measurement of the degradation products, [ 14 C] dansyl derivatives were produced, identified by autoradiography, and counted. In the degradation products in addition to histidine, the dansyl derivatives of tyrosine, phenylalanine, two leucines, alanine and lysine were identified. For comparison, glucagon was also reacted with chymotrypsin and the degradation products consisted of threonine, serine, two leucines and valine. Thus, insulin-glucagon protease degrades glucagon in a manner distinct from that of chymotrypsin.