Kinetic isotope effects are increasingly applied to investigate enzyme reactions and have been used to understand transition state structure, reaction mechanisms, quantum mechanical hydride ion tunneling and to design transition state analogue inhibitors. Binding isotope effects are an inherent part of most isotope effect measurements but are usually assumed to be negligible. More detailed studies have established surprisingly large binding isotope effects with lactate dehydrogenase, hexokinase, thymidine phosphorylase, and purine nucleoside phosphorylase. Binding reactants into catalytic sites immobilizes conformationally flexible groups, polarizes bonds, and distorts bond angle geometry, all of which generate binding isotope effects. Binding isotope effects are easily measured and provide high-resolution and detailed information on the atomic changes resulting from ligand-macromolecular interactions. Although binding isotope effects complicate kinetic isotope effect analysis, they also provide a powerful tool for finding atomic distortion in molecular interactions.