The oxidation of thiosulfate byThiobacillus thiooxidansgrown on sulfur was studied in cells, cell-free extracts, and a thiosulfate-oxidizing enzyme system. Thiosulfate was oxidized to tetrathionate by cells treated withN-ethylmaleimide with a pH optimum at 2.3. The cell-free extracts also oxidized thiosulfate with the same pH optimum and O2consumption. Untreated cells, on the other hand, oxidized thiosulfate to sulfite in the presence of 2-n-heptyl-4-hydroxyquinolineN-oxide, an inhibitor of sulfite oxidation. The cells treated withN-ethylmaleimide showed twoKmvalues for thiosulfate while the cell-free system showed only oneKm. TheKmvalue for thiosulfate generally increased with the increasing pH. A soluble thiosulfate-oxidizing enzyme system was extracted from the cells at pH 2.5 in the presence of 1 M ammonium sulfate by passage through a French pressure cell. The system contained a native cytochromecthat was reduced by thiosulfate at pH 2.5 and a thiosulfate-ferricyanide oxidoreductase activity with a pH optimum around 2.0. The acidic extract also contained a component that reduced horse heart cytochromecat a neutral pH. The reduction at an acidic pH required sulfite.Key words: thiosulfate oxidation,Thiobacillus thiooxidans, tetrathionate, sulfite.