By the use of sulfite ester substrates together with hydroxylamine as a highly reactive trapping agent, we have been able to obtain strong evidence for the intermediacy of enzyme-bound anhydride species in the pepsin-catalyzed hydrolysis of these substrates. From our observations that in the trapping experiments hydroxamate functions are introduced at the beta-carboxylates of Asp-32, Asp-215 and at least one additional Asp residue, it appears that several reactive carboxylate species can function as nucleophiles against sulfite esters, leading to the formation of anhydride species. Because the location of the hydroxamate incorporated into pepsin other than at the Asp-32 and Asp-215 residues is unknown, it remains conceivable that, at least for the action of pepsin on sulfite substrates, there are two distinct active site regions. If the possibility is considered that peptides possessing common amino-terminal residues but different acyl residues may bind productively in different fashions so that in some cases the beta-carboxylate of Asp-32 acts as the attacking nucleophile while in others the beta-carboxylate of Asp-215 acts in this way (as has been observed for sulfites), much of the confusion in the literature concerning the reactions of pepsin with peptidase may be explained.