The growth characteristics of a strain of Acidithiobacillus ferrooxidans isolated from mine water with ferrous ion, sulfur, or pyrite mineral as sole source of energy have been established. The electrokinetic behaviour of the bacterial cells was investigated and the cell surface groups were characterized by diffuse reflectance FT-IR and FT-Raman spectroscopies. The surface charge on the iron-grown cells (i.e.p., pH 2.0) is different from that on the solid-substrate-grown cells (i.e.p., pH 3.0–3.5) and the surface charge depends on the growth history of the bacteria. The FT-IR and FT-Raman spectra revealed a higher amount of protein content on the surface of the sulfur- and pyrite-grown cells compared to the iron-grown cells. These results suggest that the altered surface charge on the cells is due to differences in the protein content synthesized by bacteria exposed to different growth conditions.