A key enzyme of the thiosulfate oxidation pathway in Acidithiobacillus thiooxidans JCM7814 was investigated. As a result of assaying the enzymatic activities of thiosulfate dehydrogenase, rhodanese, and thiosulfate reductase at 5.5 of intracellular pH, the activity of thiosulfate dehydrogenase was measured as the key enzyme. The thiosulfate dehydrogenase of A. thiooxidans JCM7814 was purified using three chromatographies. The purified sample was electrophoretically homogeneous. The molecular mass of the enzyme was 27.9 kDa and it was a monomer. This enzyme had cytochrome c. The optimum pH and temperature of this enzyme were 3.5 and 35 degrees C. The enzyme was stable in the pH range from 5 to 7, and it was stable up to 45 degrees C. The isoelectric point of the enzyme was 8.9. This enzyme reacted with thiosulfate as a substrate. The Km was 0.81 mM.