The collagenolytic properties of a trypsin-like protease from the hepatopancreas of the fiddler crab Uca pugilator have been examined. All collagen types, I–V, were attacked by this enzyme. Types III and IV were degraded much more rapidly than types I, II, and V. Crab protease produced multiple cleavages in the triple helix of each collagen at 25°C; only in the case of type III collagen, however, was a major cleavage observed at a ¾:¼ locus that corresponded to the region of collagen susceptibility to mammalian collagenase action. Additionally, both the affinity and the specific activity of the crab protease for native collagen were lower than those which characterize mammalian collagenase. The results of this study, in conjunction with a previous report on the collagenolytic activity of another serine protease from the fiddler crab [Welgus, H. G., Grant, G. A., Jeffrey, J. J., & Eisen, A. Z. (1982) Biochemistry 21, 5183], suggest that the following properties distinguish the action of these invertebrate collagenolytic enzymes from the metalloenzyme collagenases of mammals: (1) broad substrate specificity, including both noncollagenous proteins and collagen types I–V; (2) ability to cleave the native triple helix of collagen at multiple loci; (3) reduced affinity or higher Km for collagen; and (4) lower specific activity on collagen fibrils.