The oxic-anoxic transition zone (OATZ) of freshwater sediments, where opposing gradients exist of reduced iron and sulfide with oxygen, creates a suitable environment for microorganisms that derive energy from the oxidation of iron or sulfide. Gradient microcosms incubated with freshwater sediment showed rapid microbial turnover of sulfide and oxygen compared with sterile systems. Microcosms with FeS as a substrate also showed growth at the OATZ and subsequent dilution series resulted in the isolation of three novel strains, of which strain J10 grows chemolithoautotrophically with reduced sulfur compounds under microaerobic conditions. All three strains are motile spirilla with bipolar flagella, related to the genera Magnetospirillum and Dechlorospirillum within the Alphaproteobacteria. Strain J10 is closely related to Magnetospirillum gryphiswaldense and is the first strain in this genus found to be capable of autotrophic growth. Thiosulfate was oxidized completely to sulfate, with a yield of 4 g protein mol(-1) thiosulfate, and autotrophic growth was evidenced by incorporation of (13)C derived from bicarbonate into biomass. A putative gene encoding ribulose 1,5-bisphosphate carboxylase/oxygenase type II was identified in strain J10, suggesting that the Calvin-Benson-Bassham cycle is used for autotrophic growth. Analogous genes are also present in other magnetospirilla, and in the autotrophically growing alphaproteobacterium magnetic vibrio MV-1.