Two strains of rod-shaped, pyrite-oxidizing acidithiobacilli, their cell envelope structure and their interaction with pyrite were investigated in this study. Cells of both strains, Acidithiobacillus ferrooxidans strain SP5/1 and the moderately thermophilic Acidithiobacillus sp. strain HV2/2, were similar in size, with slight variations in length and diameter. Two kinds of cell appendages were observed: flagella and pili. Besides a typical Gram-negative cell architecture with inner and outer membrane, enclosing a periplasm, both strains were covered by a hitherto undescribed, regularly arranged 2-D protein crystal with p2-symmetry. In A. ferrooxidans, this protein forms a stripe-like structure on the surface. A similar surface pattern with almost identical lattice vectors was also seen on the cells of strain HV2/2. For the surface layer of both bacteria, a direct contact to pyrite crystals was observed in ultrathin sections, indicating that the S-layer is involved in maintaining this contact site. Observations on an S-layer-deficient strain show, however, that cell adhesion does not strictly depend on the presence of the S-layer and that this surface protein has an influence on cell shape. Furthermore, the presented data suggest the ability of the S-layer protein to complex Fe(3+) ions, suggesting a role in the physiology of the microorganisms.