Human Met-ase-1 is a NK cell-specific member of a family of serine proteases (granzymes) that participate in target cell death inflicted by cytotoxic lymphocytes. This granzyme is predicted to cleave to the carboxyl side of long narrow hydrophobic amino acids (such as methionine), but not large, bulky hydrophobic amino acids (such as phenylalanine). To study the key structural features that confer this unusual serine protease specificity, active recombinant human Met-ase-1 was expressed in COS-7 cells. Protease assays of transfected COS-7 cell lysates provided evidence that an activation prohexapeptide normally regulates processing of this granzyme in NK cells. Recombinant human Met-ase-1 cleaved thiobenzylester substrates specifically after methionine, norleucine, or leucine residues in the primary substrate site (P1). Two key residues of human Met-ase-1, Lys179 Met (approximately chymotrypsin CHA192) and Ser201Gly (approximately CHA216), were mutated based upon a model structure derived from the crystal structure of chymotrypsin A. These mutants had reduced activity for substrate containing methionine at P1, but acquired chymase activity for phenylalanine at P1. Lys179 Met and Ser201Gly in the substrate-binding pocket of human Met-ase-1 restrict the preference of this granzyme for long narrow hydrophobic amino acids in the P1. A potential hydrogen-bonding interaction between these two residues on opposing sides of the substrate-binding pocket represents a novel molecular mechanism by which lymphocyte serine proteases might provide greater substrate specificity.