Publisher Summary This chapter explains the use of nuclear magnetic resonance (NMR) spectroscopy in studies of ion binding to hemoglobin. NMR spectroscopy offers a convenient way of measuring the binding of small ions to proteins. The basic requirements are that the ions have a magnetic nucleus that is amenable for NMR study and that the binding of the ion be accompanied by a change in an observable NMR parameter—usually the chemical shift or the relaxation rate. The chemical shift of a magnetic nucleus reflects changes in the electronic structure around the nucleus. Relaxation rates are dependent on time-varying interactions between the nucleus and the surroundings, changes in both the strength of these interactions, and the rate at which the interactions are modulated will also affect relaxation rates. Two principally different relaxation rates are defined in NMR spectroscopy: longitudinal and transverse.