To determine whether formate is involved in interspecies electron transfer between substrate-oxidizing bacteria and hydrogenotrophic microorganisms under anaerobic conditions, a syntrophic acetate-oxidizing bacterium Thermacetogenium phaeum strain PB was cocultured either with a formate/H-2-utilizing methanogen strain TM (designated as PB/TM coculture), or an H-2-utilizing methanogen strain DeltaH (designated as PB/DeltaH coculture). Acetate oxidation and subsequent methanogenesis in PB/TM coculture were found to be significantly faster than in PB/DeltaH coculture. Formate dehydrogenase and hydrogenase were both detected in strains PB and TM. H-2 partial pressures in the PB/TM coculture were kept tower (20 to 40 Pa) than those of the PB/DeltaH coculture (40 to 60 Pa) during the exponential growth phase. Formate was also detected in both PB/TM and PB/DeltaH cocultures, and the concentration of formate was maintained at a lower level in the PB/TM coculture (5 to 9 muM) than in the PB/DeltaH coculture. Thermodynamic calculations revealed that the concentrations of both H-2 and formate severely affect the syntrophic oxidation of acetate. These results strongly indicate that not only H-2 but also formate may be involved in interspecies electron transfer.