Pepsin reacts stoicheiometrically with the active-site-directed irreversible inhibitor N-diazoacetyl-l-phenylalanine methyl ester, with concomitant loss of all proteolytic and peptidolytic activity. The reagent esterifies a unique aspartic acid residue in pepsin, which is in the sequence:Ile-Val-Asp-Thr-Gly-Thr-Ser