We isolated several thermotolerant Acetobacter species of which MSU10 strain, identified as Acetobacter pasteurianus, could grow well on agar plates at 41A degrees C, tolerate to 1.5% acetic acid or 4% ethanol at 39A degrees C, similarly seen with A. pasteurianus SKU1108 previously isolated. The MSU10 strain showed higher acetic acid productivity in a medium containing 6% ethanol at 37A degrees C than SKU1108 while SKU1108 strain could accumulate more acetic acid in a medium supplemented with 4-5% ethanol at the same temperature. The fermentation ability at 37A degrees C of these thermotolerant strains was superior to that of mesophilic A. pasteurianus IFO3191 strain having weak growth and very delayed acetic acid production at 37A degrees C even at 4% ethanol. Alcohol dehydrogenases (ADHs) were purified from MSU10, SKU1108, and IFO3191 strains, and their properties were compared related to the thermotolerance. ADH of the thermotolerant strains had a little higher optimal temperature and heat stability than that of mesophilic IFO3191. More critically, ADHs from MSU10 and SKU1108 strains exhibited a higher resistance to ethanol and acetic acid than IFO3191 enzyme at elevated temperature. Furthermore, in this study, the ADH genes were cloned, and the amino acid sequences of ADH subunit I, subunit II, and subunit III were compared. The difference in the amino acid residues could be seen, seemingly related to the thermotolerance, between MSU10 or SKU1108 ADH and IFO 3191 ADH.