We used Mössbauer spectroscopy to study the iron content, the redox state, and the binding site of iron in substantia nigra (SN) from parkinsonian (PD) and control brains. Measurements performed on fresh-frozen, formalin-fixed, and lyophilized samples demonstrated the presence of ferric (Fe3+) iron only, both in PD and control SN. Ferrous iron, if present at all, may represent at most 5% of the total iron. We found no difference in the total amount of iron in SN between PD and control brains. The Mössbauer spectra observed at 4.1 K in fresh (frozen or lyophilized) samples were different from those obtained in formalin-fixed (frozen or lyophilized) samples. In the fresh samples, only ferritin-like iron was observed, whereas in the samples frozen or lyophilized from formalin, non-ferritin iron was detected.