(1)α-Deuterium kinetic isotope effects on Vmax. for hydrolysis of both β-D-glucopyranosylpyridinium ions and aryl β-D-glucopyranosides are in the range kH/kD 1.08–1.14, indicating that bond-breaking limits the rate of hydrolysis of both sets of substrates. The variation of kcat with aglycone acidity, expressed by log kcat=A+βlgpKa, is governed by a βlg value of –0.96 ± 0.19 for the N-glycosides and –0.05 ± 0.05 for the O-glycosides. The latter, low value of |βlg| is evidence for extensive proton donation to the oxygen at the transition state, even for the departure of acidic aglycones. The dependence of rate on protonation of a group of pKa < 6 required by this idea is indeed observed. (2) Comparison of log kcat values with spontaneous hydrolysis rates indicates that nucleophilic and non-covalent interactions accelerate C–N bond cleavage in the ES complex by a factor of 10(8 + 0.3[pka]N) for glucosyl pyridinium salts where [pKa]N is the pKa of the pyridine. On the assumption that these interactions are similar for O-glucosides, proton donation to the aglycone oxygen atom can be estimated to contribute a rate-enhancement of 10(0.1 + 0.7[pKa]O), where [pKa]O is that of the free phenol. (3)D-Glucono-δ-lactone and 5-amino-5-deoxy-D-gluconolactam are bound 102.8 and 102.2 times, respectively, more tightly than β-D-glucopyranose, because of their analogy to a transition state in which α-deuterium kinetic isotope effects have shown the anomeric carbon atom to have substantial sp2 character. (4) Cationic inhibitors are bound 102.5–103.5 times more tightly than directly comparable neutral ones, if the positive charge resides on the equatorial substituent at C-1, but protonated 2-amino-2-deoxyglucose is bound no more tightly than glucose. This indicates the presence of a negative charge in the El complex near C-1 on the α face of the pyranose ring (an aspartate residue previously identified by covalent labelling) which is partly compensated by a positive charge near C-2.