When incubated under anaerobic conditions, five strains of Thiobacillus ferrooxidans tested produced hydrogen sulfide (H2S) from elemental sulfur at pH 1.5. However, among the strains, T. ferrooxidans NASF-1 and AP19-3 were able to use both elemental sulfur and tetrathionate as electron accepters for H2S production at pH 1.5. The mechanism of H2S production from tetrathionate was studied with intact cells of strain NASF-1. Strain NASF-1 was unable to use dithionate, trithionate, or pentathionate as an electron acceptor. After 12 h of incubation under anaerobic conditions at 30 degrees C, 1.3 mu mol of tetrathionate in the reaction mixture was decomposed, and 0.78 mu mol of H2S and 0.6 mu mol of trithionate were produced. Thiosulfate and sulfite were not detected in the reaction mixture. From these results, we propose that H2S is produced at pH 1.5 from tetrathionate by T. ferrooxidans NASF-1, via the following two-step reaction, in which AH(2) represents an unknown electron donor in NASF-1 cells. Namely, tetrathionate is decomposed by tetrathionate-decomposing enzyme to give trithionate and elemental sulfur (S4O62--->S3O62-+S-0, Po. 1), and the elemental sulfur thus produced is reduced by sulfur reductase using electrons from AH(2) to give H2S (S-0 + AH(2)-->H2S + A, Eq. 2). The optimum pH and temperature for H2S production from tetrathionate under argon gas were 1.5 and 30 degrees C, respectively. Under argon gas, the H2S production from tetrathionate stopped after Id of incubation, producing a total of 2.5 mu mol of H2S/5 mg protein. In contrast, under H-2 conditions, H2S production continued for 6 d, producing a total of 10.0 mu mol of H2S/5 mg protein. These results suggest that electrons from H-2 were used to reduce elemental sulfur produced as an intermediate to give H LS. Potassium cyanide at 0.5 mM slightly inhibited H2S production from tetrathionate, but increased that from elemental sulfur 3-fold. 2,4-Dinitrophenol at 0.05 mM, carbonylcyanide-m-chlorophenyl-hydrazone at 0.01 mM, mercury chloride at 0.05 mM, and sodium selenate at 1.0 mM almost completely inhibited H2S production from tetrathionate, but not from elemental sulfur.