Isotopic substitution is known to affect kinetic rate constants and equilibrium constants in chemistry. In this study, we have used tritium substitution and high pH to probe the glucose reversible arrow glucose(-) + H+ equilibrium. Passing partially ionized mixtures of [H-3]- and [C-14]glucose over anionic exchange resin has permitted the detection of subtle differences in pK(a). We have found that, at pH 11.7 in an anionic exchange system, [3H]glucose always elutes ahead of the [14C]glucose, and we report isotope effects of 1.051 +/- 0.0007, 1.012 +/- 1.0005, 1.014 +/- 0.0004, 1.024 +/- 0.0003, 1.014 0.0004, and 1.015 +/- 0.0014 for [1-H-3]-, [2-H-3]-, [3-H-3]-, [4-H-3]-, [5-H-3]-, and [6,6-H-3(2)]glucose, respectively, as compared to either [2-C-14]- or [6-C-14]glucose. The elevated isotope effects at H1 and H4 imply unusual charge sharing in anionic aqueous glucose. Base titration of C-13-chemical shift changes demonstrates the dominance of pyranose forms at elevated pH, and ab initio isotope effect computations on gas-phase glucose anions are presented.