H-1 NMR chemical shift assignments were established for (NH)-H-delta 1 (16.9 ppm) and (NH)-H-epsilon 2 (16.1 ppm) of the active-center His57 for the complex of MeOSuc-Ala-Ala-Pro-boroPhe (BoroPhe) with chymotrypsin and for the (CH)-H-epsilon 1 proton (9.2 ppm at low pH and 8.5 ppm at high pH) of His57 in uncomplexed chymotrypsin. The assignment for (CH)-H-epsilon 1 corrects previous assignments and reveals an unusual environment of this carbon-bound proton. The relative NH assignments are reversed from the order of NH assignments previously found for alpha-lytic protease complexes with boronate inhibitors. Isotopic fractionation factors (H/D) were determined using H-1 NMR for hydrogen bonds to the active site histidine in BoroPhe complexes with chymotrypsin and subtilisin E, and for uncomplexed chymotrypsin. Measured fractionation factors accurate to about +/-0.1 were 0.82 (pH 10) and 0.64 (pH 3) for the (NH)-H-delta 1 proton of uncomplexed chymotrypsin. In the presence of BoroPhe at pH 6.5, the (NH)-H-delta 1 fractionation factors were 0.65 for the chymotrypsin-inhibitor complex, and 0.53 for the subtilisin-inhibitor complex. Measurements for the (NH)-H-epsilon 2 fractionation factor were 1.05 (uncomplexed chymotrypsin at pH 10), 0.93 (BoroPhe-chymotrypsin at pH 6.5), and 0.76 (BoroPhe-subtilisin at pH 6.5). Both model calculations of isotopic fractionation factors and experimentally determined inhibition constants were used in the analysis of the fractionation-factor results.