The rates of thiosulfate, elemental sulfur (S0) and sulfite oxidation were measured respirometrically with an oxygen electrode using young cells of Thiobacillus versutus growing chemolithoautotrophically on thiosulfate under normal air pressure. Myxothiazol, an inhibitor of the cytochrome b−c1 segment, and HQNO (2-N-heptyl-4-hydroxyquiniline N-oxide), acting in the quinone-cytochrome b region, both significantly inhibited the thiosulfate oxidation rate. The effect on the oxidation rate of S0 was even stronger. The oxidation of sulfite or ascorbate + TMPD (N,N,N′,N′-tetramethyl-p-phenylenediamine) (substrates releasing electrons at the level of cytochrome c) was not inhibited by myxothiazol and HQNO. Thiosulfate, S0, sulfite and ascorbate + TMPD oxidations were strongly inhibited by KCN. These respiratory activities were almost completely eliminated by cell breakage. The reduction of b-type cytochrome was observed in thiosulfate-reduced minus sulfite-reduced difference spectra. This study confirms that S0 is an important intermediate of thiosulfate oxidation in Thiobacillus versutus, and that electrons released by S0 oxidation enter the respiratory chain in the quinone-cytochrome b region. This would allow an increased gain of energy, while less energy would probably be required for pyridine-nucleotide reduction.