Cytochrome c' from Rhodospirillum rubrum has been studied by proton magnetic resonance (NMR) at 270 MHz. The pH and temperature-dependence properties as well as proton water relaxation enhancement and bulk susceptibility measurements were examined. We conclude that the fifth ligand to the iron is histidine. The pH-dependent shift of the heme methyl resonances of the ferric protein shows pKa's at 5.8 and 8.7. The low-pH equilibrium causes only minor changes in the properties of the protein. However, the high-pH equilibrium causes large changes throughout the NMR spectra which correlate with the reported visible spectral changes. These NMR spectral changes are compared with the low-temperature EPR and Mössbauer spectroscopic data. Analyses of the NMR data show that a second histidine, which is present in the sequence of c' from R. rubrum but is not conserved in other cytochromes c', is not a "distal" histidine. The nature of the sixth ligand and the significance of the high-pH transition are discussed.