The kinetics of 14 peptide substrates of carboxypeptidase A have been studied for the purpose of evaluating P 1 –P 3 /S 1 –S 3 interactions. It was found that the amide group at P 1 –P 2 is required for efficient catalysis. This observation is consistent with previously proposed hydrogen bonding interactions, based on crystallographic data, between the P 1 NH and Tyr-248 and between the P 2 carbonyl oxygen and Arg-71. In contrast, substitution of the benzamido amide group (at P 2 –P 3 ) ofN-benzoylglycylglycyl-L-phenylalanine by –CH 2 CH 2 – resulted in more effective catalysis. In this case hydrophobic interactions are important in the ground state and in the transition state of the rate-determining step.