The proton chemical shift range 13-19 ppm downfield from silanes was reexamined for aqueous solutions of chymotrypsin in the absence and presence of boronic acid inhibitors. While B-11 NMR experiments had conclusively demonstrated that these inhibitors formed a tetrahedral complex at the active center and that Ser195 was required for the binding of these putative transition state analogs (Zhong, S.; Jordan, F.; Kettner, C.; Polgar, L. J. Am. Chem. Sec. 1991, 113, 9429-9435), there is important additional information available from observation of His57 imidazole NH proton resonances, clearly demonstrating the conditions under which such complexes exist. Supported by the assignments made for alpha-lytic protease (Bachovchin, W. W.; Wong, W. Y. L.; Farr-Jones, S.; Shenvi, A. B.; Kettner, C. A. Biochemistry 1988, 27, 7689-7697), information is presented on differential behavior of the HNdelta 1 and (NH)-H-epsilon 2 protons, depending on the strength of inhibition. Several complexes of a very potent, slow-binding inhibitor, MeO-Suc-Ala-Ala-Pro-BoroPhe, can be observed, some at strongly alkaline and acid conditions, whereas only one complex is observed with the rapidly reversible inhibitor MeO-Suc-Ala-Ala-Pro-BoroVal. At intermediate pH values, both of these inhibitors (a) must be bound to Ser195, since resonances corresponding to both HNdelta 1 and (NH)-H-epsilon 2 are clearly detectable, and (b) form complexes characterized by the same chemical shifts for their HNdelta 1 and (NH)-H-epsilon 2 resonances, the principal differences being reflected in the exchange rates of these protons, presumably due to their differential accessibility to water. At intermediate pH, the HNdelta 1 and (NH)-H-epsilon 2 renonances have pH-independent chemical shifts, providing evidence for an elevated pK of His57 on formation of the negatively charged boronates, as expected; the pK elevation parallels the strength of inhibition. While the peptideboronic acids appear to give rise to only one type of complex with chymotrypsin at intermediate pH values, 3,5-bis(trifluoromethyl)phenylboronic acid gives rise to two complexes, in accord with B-11 NMR studies, one Ser195, and the other probably His57-bound.