The sulfur-binding protein of Thiobacillus ferrooxidans ATCC 23270 was investigated. The protein composition of the bacterium's cell surface changed according to the culture substrate. Sulfur-grown cells showed greater adhesion to sulfur than iron-grown cells. The sulfur-grown cells synthesized a 40-kDa surface protein which was not synthesized by iron-grown cells. The 40-kDa protein had thiol groups and strongly adhered to elemental sulfur powder. This adhesion was not disturbed by Triton X-100, which can quench hydrophobic interactions. However, adhesion was disturbed by 2-mercaptoethanol, which broke the disulfide bond. The thiol groups of the 40-kDa protein formed a disulfide bond with elemental sulfur and mediated the strong adhesion between T. ferrooxidans cells and elemental sulfur. The 40-kDa protein was located on the flagella. The location of the protein would make it possible for cells to be in closer contact with the surface of elemental sulfur powder.