Mössbauer spectroscopy was used to study the tetraheme cytochrome c3 from Desulfovibrio baculatus (DSM 1743). Samples with different degrees of reduction were prepared using a redoxtitration technique. In the reduced cytochrome c3, all four hemes are reduced and exhibit diamagnetic Mössbauer spectra typical for low-spin ferrous hemes (S = 0). In the oxidized protein, the hemes are low-spin ferric (S = 1/2) and exhibit overlapping magnetic Mössbauer spectra. A method of differential spectroscopy was applied to deconvolute the four overlapping heme spectra and a crystal-field model was used for data analysis. Characteristic Mössbauer spectral components for each heme group are obtained. Hyperfine and crystal-field parameters for all four hemes are determined from these deconvoluted spectra.