Extracellular polymeric substances (EPS) of Acidithiobacillus ferrooxidans and other leaching microorganisms mediate the attachment of cells to pyrite and other minerals. They also play a pivotal role in indirect leaching of base and precious metals via the contact mechanism. The aim of this study is to get more insight on the influence of the growth substrates iron(II) ions, pyrite, chalcopyrite and elemental sulfur on EPS formation, attachment and biofilm formation. The synthesis of EPS by cells of A. ferrooxidans strain A2 is strongely influenced by the growth substrate or attachment substratum of the cells. Cells grown with soluble iron(II) ions generally generate less EPS than cells grown with solid pyrite, chalcopyrite or elemental sulfur. Planktonic cells grown in the presence of solid substrate produce two to four times more EPS than iron(II) ion grown cells. With sessile cells, this factor is further increased to 50 to 240 depending on the specific substrate. The EPS of all the differently grown planktonic and sessile cells of A. ferrooxidans strain A2 contained neutral sugars, fatty acids, uronic acids, proteins and metal ions. The composition of these compounds varied with the growth substrate and type (planktonic or sessile). The attachment behavior of cells of A. ferrooxidans strain A2 also differed with the substrate of the pre-culture. Cells grown on iron(II) ions, pyrite or chalcopyrite attached rapidly to pyrite and chalcopyrite, while attachment to elemental sulfur was poor. On the contrary, sulfur grown cells attached well to elemental sulfur but weakly to pyrite and chalcopyrite. Attachment of EPS-free cells to all substrates was also diminished. Cells of A. ferrooxidans strain A2 cover mineral surfaces with a dense biofilm after a few days of cultivation, as visualized by fluorescence microscopy and AFM. Large amounts of EPS are formed, which eventually cover the cells and the mineral surface. Even after a few weeks of cultivation the biofilm remained monolayered on all substrates.