A number of strains of heterotrophic bacteria were isolated from various environments on the basis of their potential to oxidize inorganic sulfur compounds to tetrathionate. The isolates were screened for the ability to oxidize thiosulfate under denitrifying conditions. Many of them could grow anaerobically with acetate and nitrate, and eight strains could oxidize thiosulfate to tetrathionate under the same conditions. In batch cultures with acetate as carbon and energy source, most active anaerobic thiosulfate oxidation occurred with N2O as electron acceptor. The level of anaerobic thiosulfate-oxidizing activity in cultures and cell suspensions supplied with nitrate correlated with the activity of nitrite reductase in cell suspensions. Some strains converted thiosulfate to tetrathionate equally well with nitrite, nitrate and N2O as electron acceptors. Others functioned best with N2O during anaerobic thiosulfate oxidation. The latter strains appeared to have a lower level of nitrite reductase activity. Thiosulfate oxidation under anaerobic conditions was much slower than in the presence of oxygen, and was obviously controlled by the availability of organic electron donor. The strains had DNA-DNA similarity levels higher than 30%. Sequence analysis of the 16S rRNA gene of four selected isolates showed their affiliation to specific genomovars of Pseudomonas stutzeri and the proposed new species, Pseudomonas balearica. As shown by 16S rRNA sequence analysis and DNA-DNA hybridization, the previously misnamed ‘Flavobacterium lutescens’ (ATCC 27951) is also a P. stutzeri strain which can oxidize thiosulfate to tetrathionate aerobically and anaerobically in the presence of N2O. The data suggest that tetrathionate-forming heterotrophic bacteria, in particular those belonging to the P. stutzeri‘superspecies’, can play a much more significant role in the biogeochemical cycles than was previously recognized.