A thiosulfate reductase from Desulfotomaculum nigrificans has been partially purified by ammonium sulfate fractionation, diethylaminoethylcellulose chromatography, and sucrose density gradient centrifugation. With inner-and outer-labeled (35)S-thiosulfate, the enzyme reduced only the outer sulfur atom to hydrogen sulfide. The enzyme was inhibited by sulfite and also by several sulfhydryl inhibitors. The K(m) value for this enzyme was calculated to be 1.3 x 10(-1)m. Other inorganic sulfur compounds, such as sulfate, sulfite, tetrathionate, and dithionate, were not reduced by this enzyme.