Lactobacillus acetotolerans was isolated from a turbid fermentation broth of rice vinegar and showing strong acetic acid tolerance. The effect of acetate concentration in the cultivation media to the cellular fatty acid composition was examined. The cellular fatty acids of L. acetotolerans grown in MRS medium were consisted of hexadecenoic acid, octadecenoic acid, octadecanoic acid and cyclopropane octadecanoic acid as the typical lactic acid bacteria. The cells grown in MRS medium containing 3% sodium acetate contained more than 50% of an unknown fatty acid. The unknown fatty acid was identified as 10OH-octadecanoic acid by gas chromatography mass spectrometry with the reference materials. 10OH-Octadecanoic acid was found only when L. acetotolerans was cultivated with more than 3% sodium acetate and 0.1% Tween 80 in the medium.