The term chymase is used to signify a chymotrypsin-like protease stored within the secretory granules of mast cells. Primarily based on amino acid sequence homology, 18 chymases have been identified among different animals. This study, which compares the structure of the primary specificity pocket (S1 subsite), defines a subgroup of four chymases likely to have a substrate specificity with more elastase- than chymotrypsin-like qualities. This difference is due, primarily, to finding a Val instead of a Gly at residue 199, a position corresponding to Gly216 in bovine chymotrypsin and Val216 in neutrophil and porcine elastases. Chymases with Val at 199 are found only in animals expressing multiple chymases, consistent with the premise that their substrate specificity differs from that of chymases with Gly at 199.