Among 150 pure strains of iron-oxidizing bacteria obtained from natural environments, two strains, Funis 2-1 and OK1-50, had the ability to use potassium tetrathionate (K2S4O6) as a sole energy source for growth. Funis 2-1 was a gram-negative, rod-shaped, acidophilic iron- and sulfur-oxidizing chemolithotrophic bacterium and had the same cytochrome composition and mean G+C content of DNA as Thiobacillus ferrooxidans, indicating that the strain is T. ferrooxidans. A tetrathionate-decomposing enzyme that catalyzes the disproportionate metabolism of 4 mol of tetrathionate into 7 mol of thiosulfate and 2 mol of sulfate was located on the plasma membrane of K2S4O6-grown, but not Fe2+-grown Funis 2-1 cells. Washed intact cells and cell extracts prepared from Funis 2-1 cells grown on K2S4O6 medium supplemented,vith more than 11 mM FeSO4 did not show K2S4O6-decomposing enzyme activity. K2S4O6-decomposing enzyme was purified to homogeneity from K2S4O6-grown Funis 2-1 cells. The apparent molecular weight of this enzyme was estimated to be 50,000 by gel filtration, 50,000 by SDS-PAGE, and 49,600 using a time-of-flight mass spectrometer, indicating that the enzyme is monomeric. The enzyme was most active at pH 3.5 and 50 degrees C and the activity was enhanced approximately 18 fold by a concentration of 200 mM of sulfate ion. The Michaelis constant of this enzyme for K2S4O6 was 0.73 mM.