Kinetic constants are reported for alpha-chymotrypsin- and Streptomyces griseus protease 3 (SGP3)-catalyzed amide hydrolysis of a number of peptide amides of varying substrate chain length. alpha-Chymotrypsin, but not SGP3, will hydrolyze rapidly specific acetyl amino acid amides. SGP3-catalyzed, but not alpha-chymotrypsin-catalyzed, hydrolysis is greatly stimulated by the presence of up to four amino acid residues N-terminal to the scissile bond of the substrate. The enzyme-substrate interactions utilized to promote hydrolysis, therefore, differ in these two enzymes, which, in other respects, show marked similarities. alpha-Chymotrypsin depends mainly on primary enzyme-substrate contacts, those with the amino acid residue (P1) whose carbonyl group forms part of the scissile bond, whereas SGP3 depends mainly on "secondary" enzyme-substrate contacts with amino acid residues (P2-P4) more remote from the scissile bond. A comparison with porcine elastase, a related serine protease, indicates that there is an inverse relation between the importance of primary and secondary enzyme substrate interactions in this family of enzymes. A rationale is proposed for this effect based on the observation that both types of enzyme-substrate interaction predominantly affect the rate constant for the acylation step of substrate hydrolysis.