A novel, anaerobic, non-spore-forming, mobile, Gram-negative, thermophilic bacterium, strain TMOT, was isolated from a thermophilic sulfate-reducing bioreactor operated at 65 C with methanol as the sole substrate. The G+C content of the DNA of strain TMOT was 39.2 mol%. The optimum pH, NaCl concentration, and temperature for growth were 7.0, 1.0%, and 65 degrees C, respectively. Strain TMOT was able to degrade methanol to CO2 and H2 in syntrophic culture with Methanothermobacter thermautotrophicus AH or Thermodesulfovibrio yellowstonii. Thiosulfate, elemental sulfur, Fe(III) and anthraquinone-2,6-disulfonate were able to serve as electron acceptors during methanol degradation. In the presence of thiosulfate or elemental sulfur, methanol was converted to CO2 and partly to alanine. In pure culture, strain TMOT was also able to ferment methanol to acetate, CO2 and H2. However, this degradation occurred slower than in syntrophic cultures or in the presence of electron acceptors. Yeast extract was required for growth. Besides growing on methanol, strain TMOT grew by fermentation on a variety of carbohydrates including monomeric and oligomeric sugars, starch and xylan. Acetate, alanine, CO2, H2, and traces of ethanol, lactate and alpha-aminobutyrate were produced during glucose fermentation. Comparison of 16S rDNA genes revealed that strain TMOT is related to Thermotoga subterranea (98%) and Thermotoga elfii (98%). The type strain is TMOT (= DSM 14385T = ATCC BAA-301T). On the basis of the fact that these organisms differ physiologically from strain TMOT, it is proposed that strain TMOT be classified as a new species, within the genus Thermotoga, as Thermotoga lettingae.