Mössbauer spectroscopy has been used to study three non-heme iron proteins. The spectra of the ferredoxins from Chromatium and from spinach show similar changes on oxidation and reduction. In confirmation of chemical experiments, only one of the two irons in spinach ferredoxin is found to be reduced by sodium dithionite. The Mössbauer spectra of oxidized high-potential iron protein from Chromatium show that the electronic configurations of the four iron atoms are equivalent and are all affected by unpaired electron spins giving rise to broad, diffuse Mössbauer spectra in the temperature range from 60 to 4.6°K. The Mössbauer spectrum of reduced high-potential iron protein is distinctly different from those of the two reduced ferredoxins, and further justifies regarding high-potential iron protein as a non-heme iron protein distinct from the ferredoxin class. © 1968, American Chemical Society. All rights reserved.