The proteolytic specificity of cathepsin L on glucagon was determined. Major cleavages are found between Thr7 and Ser8, Asp15 and Ser16, and between Met27 and Asn28. The bonds Ser11-Lys12, Val23-Gln24, and Gln24-Trp25 are hydrolyzed to a relatively low extent only. Whereas cathepsin B hydroxyzes glucagon at the C-terminus by a peptidyldipeptidase mechanism, cathepsin L cleaves the same substrate clearly as endopeptidase.