The action of α-chymotrypsin onL- andD-phenylalanine ethyl esters (PEE),L- andD-phenylalaninep-nitrobenzyl esters (PNBE),L-phenylalanine methyl and isopropyl esters, andN-methyl-L-phenylalamne methyl ester has been studied using a pH-stat. TheD-esters were not hydrolyzed but acted as competitive inhibitors of the hydrolysis of theL-isomers. TheN-methyl ester was very slowly hydrolyzed due to its lowKcat. ForL-PEE (pK7.23) andL-PNBE (pK6.93), the activity of α-chymotrypsin is displaced to a more acid region relative to that for theN-acyl amino acid esters. TheKmincreases sharply below pH 6.5 while thekcatandkcat/Kmshow maxima at pH 6 and 7.6, respectively. On the acid sidekcatis controlled by a basic group of pK4.86 forL-PNBE and pK5.1 forL-PEE, andkcat/Kmby a basic group of pK6.4 forL-PNBE and pK6.6 forL-PEE. It is proposed that (i) deacylation is rate-limiting, (ii) in the catalytically active entities of the enzyme–substrate complex and acyl enzyme, the α-amino group of the substrate is protonated, (iii) the pKof the basic group on the acyl enzyme is considerably lowered by the presence of theof the substrate, and (iv) the increase inKmandKibelow pH 6.8 is due to the development of unfavorable charge interactions.