The amino acid sequence of a collagenolytic protease from the hepatopancreas of the fiddler crab, Uca pugilator, was determined from the structures of overlapping tryptic, chymotryptic, thermolytic, staphylococcal protease, and cyanogen bromide peptides together with automated sequencer analysis of the intact protein. Crab collagenase is a serine protease composed of 226 residues which is capable of degrading the native triple helix of collagen under physiological conditions. When aligned for optimal homology, crab collagenase displays 35% identity with bovine trypsin, 38% with bovine chymotrypsin B, and 32% with porcine elastase. The six half-cystinyl residues in crab collagenase correspond to those forming three of the five disulfide bonds in chymotrypsin. The residues forming the charge relay system of the active site of chymotrypsin (His-57, Asp-102, and Ser-195) are found in corresponding regions in crab collagenase, and the sequences around these residues are well conserved. The primary structure of crab collagenase is the first reported for a serine protease from crustacean hepatopancreas and the first reported for a serine protease possessing the unusual property of being able to degrade native helical collagen.