Treatment of swine pepsin with acetylimidazole to acetylate approximately five of its 16 tyrosyl residues causes a significant enhancement of catalytic efficiency (kcat/Km) toward substrates such as dansyl-glycyl-glycyl-L-phenylalanyl-L-phenylalanine 3-(4-pyridyl)propyl ester and benzyloxy-carbonyl-(glycyl)n-p-nitroLphenylalnyl-Lphenylalanyl-L-tyrosine (where n = 0, 1,2). Stopped-flow kinetic studies, under conditions of enzyme excess, with the dansyl peptide have shown that, as with untreated pepsin, the rate-limiting step in the over-all catalytic process is associated with the decomposition of the first detectable enzyme-substrate complex, whose dissociation constant is approximately equal to the Km found in steady-state kinetic experiments. With substrates of the type benzoyl-(glycyl)n-nitro-L-phenylalanyl-L-tyrosine, an increase in the chain length of the peptide leads to an increase in the value of kcat/Km, supporting the view that secondary enzyme-substrate interactions may produce at the extended active site conformational changes that are reflected in higher catalytic efficiency. This effect is more marked with acetyl-pepsin than with untreated pepsin, and suggests that the conformational mobility of the active site is increased by partial acetylation. Acetyl-pepsin is less effective than untreated pepsin in catalyzing transpeptidation reactions in which acetyl-L-phenylalanyl-L-tyrosine and benzyloxycarbonyl-(glycyl)n-p-nitro-L-phenylalanine are the reactants; this finding is consistent with the more rapid hydrolysis of the product of transpeptidation.