We have utilized tritium isotope effects to probe the in vitro binding equilibrium between glucose and human brain hexokinase (E.C.2.7.1.1). Replacing a backbone hydrogen atom in glucose with tritium can significantly increase or decrease the equilibrium association constant. Specifically, the equilibrium tritium isotope effects are 1.027 +/- 0.002, 0.927 +/- 0.0003, 1.027 +/- 0.004, 1.051 +/- 0.001, 0.988 +/- 0.001, and 1.065 +/- 0.003 for [1-f]-, [2-f]-, [3-f]-, [4-f]-, [5-f]-, and [6,6-t(2)]glucose, respectively. We have shown that the existence of prebinding equilibrium isotope effects can contribute to binding isotope effect studies but that this effect is insignificant for glucose binding to hexokinase. The binding isotope effects are interpreted in the context of structural studies of hexokinase-glucose complexes. Ab initio calculations on 2-propanol with or without a hydrogen bonding partner, in steric collision with formaldehyde or methane, and on ethanol, cyclohexanol and 1-hydroxymethyl-tetrahydropyran are presented to clarify the magnitude of isotope effects possible in such interactions and the accompanying changes in free energy. Position-specific binding isotope effects provide direct evidence of the partial deprotonation and activation of 06 by Asp657, of other hydrogen bonding interactions with ionic residues, and of the steric compression of CH2 by the backbone carbonyl of Ser603.