A number of peptides of the general formula Ac-Pro-Ala-X-Phe-NH2, where X = Gly, Ala, Leu, Phe or Pro, have been synthesized for the study of S2-P2 enzyme-substrate interactions in three serine proteases: alpha-chymotrypsin and Streptomyces griseus proteases 1 and 3. All three enzymes interacted favorably with those peptides with hydrophobic, non-aromatic, P2 amino acid residues, leucine being optimal. The increase in kcat/Km on going from the P2 glycine to the P2 leucine peptide was 30-fold in alpha-chymotrypsin and greater than 150-fold and 350-fold in the Streptomyces enzymes, being due to both increased affinities and higher acylation rates. Hydrophobic S2-P2 interactions, particularly in S. griseus proteases 1 and 3, appear to be the most important enzyme-substrate interactions apart from S1-P1. The kinetic data is discussed in relation to the tertiary structures of the active centers of the enzymes.