Studies on the specificity of chymotrypsin have demonstrated that the enzyme hydrolyzes peptide linkages of tyrosyl and phenylalanyl peptides which involve the carbonyl residue of the aromatic amino acids (1). Subsequent studies have shown that this enzyme hydrolyzes the ester bond of acylamino acid ester having general formula R′CO-NH(CHR)CO-OR″. In the substrates tested, the group R′CO- was acetyl or benzoyl for example, admittedly unphysiological substituents (1). No acylamino acid ester in which the acyl substituents are amino acid goups have been reported to be subjected to the action of chymotrypsin. In the preceding paper of this series, a number of aminoacyl-L-tyrosinamide have been tested as substrates for chymotrypsin (2). In the present study, a number of aminoacyl-L-tyrosine ethyl esters in which the aminoacyl substituents are the aliphatic amino acid groups were prepared, and were subjected to the action of α-chymotrypsin.