The downfield histidine imidazole N proton resonances of bovine superoxide dismutase [Lippard, S. J., Burger, A. R., Ugurbil, K., Pantoliano, M. W., & Valentine, J. S. (1977) Biochemistry 16, 1136] have been studied by pulsed NMR in H2O buffer. Exchange rates with solvent and nuclear Overhauser effects (NOE), mainly with imidazole C2 and C4 protons on the same residue, have been found. In the reduced state, resonances observed (at pH 7) at 13.9 and 12.8 ppm downfield from DSS are shown to be His-41 N3 and N1 protons, respectively, by NOE to a common C2-H resonance previously identified with this residue [Cass, A. E. G., Hill, H. A. O., Smith, B. E., Bannister, J. V. & Bannister, W. H. (1977) Biochemistry 16, 3061] and by mutual second-order NOE. The 12.8-ppm resonance shifts upfield above pH 9.5, showing that His-41 has a pka of about 10.4. Protons resonating at 15.35 and 13.4 ppm are the most slowly exchanging, with similar rates (~2 X 10-s s-1 at 37 °C) and activation energies (~30 kcal/mol). They are identified with the two most buried active-site histidine residues, 44 and 69, and are both N1 coordinated to the active-site metals. The NOE of the resonance at 12.5 ppm shows that the corresponding residue is N3 coordinated. The resonance at 13.9 ppm and a broad resonance at 12.6 ppm show base-catalyzed kinetic broadening above neutral pH; the other resonances remain observable to pH 10.5. In the oxidized state, NOE established that broad resonances at 12.1 and 13.6 ppm originate from His-41. The magnitude of the NOE is used to estimate several interproton distances, and strategies for selective NOE observation are discussed. © 1979, American Chemical Society. All rights reserved.