A soluble c-type cytochrome was purified to homogeneity from Thiobacillus ferrooxidans. This cytochrome is characterised by an alpha-peak wavelength of 552 nm, a molecular mass of 21 193 Da (as determined by mass spectroscopy), and a pI value of 9. N-terminal sequencing yielded the polypeptide sequence up to the 50th residue. The iron content of 1.9 Fe/molecule and the heme/molecule ratio of 2.15 identified this cytochrome as a diheme protein. Optical redox titrations at pH 3.0 revealed the presence of two distinguishable redox species with Em = 385 mV +/- 20 mV and Em = 480 mV +/- 20 mV. EPR spectra recorded on this heme protein showed the presence of two distinct spectral species with gz = 3.1 and gz = 3.35. The gz = 3.35 heme corresponds to the higher potential redox species. In line with the differences in Em values, the two heme species were oxidised by O2 with significantly differing half-times. All the above mentioned properties demonstrate that this heme protein belongs to the c4 family of diheme cytochromes. The characteristics and functional role of the studied heme protein are discussed with reference to other c-type cytochromes described in Thiobacilli. Its properties are furthermore compared to other members of the cytochrome c4 family.