The Mössbauer spectra of horseradish peroxidase and its peroxide derivatives have been studied. Optical spectra of frozen solutions prepared under the same conditions as the Mössbauer samples have been used to confirm that concentrated frozen samples of compounds I and II were obtained in nearly pure form. The data show that there is a major change in iron electronic configuration on going from the resting enzyme to the primary or secondary compounds (compounds I and II) formed when the protein interacts with peroxide. This change is compatible with the formation of an Fe(IV) configuration. There is no major change in Mössbauer spectra on going from compound I to II, so that the extra oxidizing equivalent of compound I is probably not found in a Fe(V) configuration, but instead must be localized at a site other than that of the iron itself. © 1969, American Chemical Society. All rights reserved.