The effects of 3 fermentation temperatures (30, 37, and 42°C) and 3 fermentation pH (4.5, 5, and 6) on the cryotolerance of Lactobacillus acidophilus RD758 were studied in relation to their fatty acid composition. Cryotolerance was defined as the ability of the cells to recover their acidification activity after freezing and frozen storage at -20°C. Better cryotolerance was obtained in cells grown at 30°C or at pH 5; these cells showed no loss in acidification activity during freezing and a low rate of loss in acidification activity during frozen storage. On the other hand, cells grown at 42°C or at pH 4.5 displayed poor cryotolerance. The membrane fatty acid composition was analyzed and related to the cryotolerance using principal component analysis. The improved cryotolerance observed during the freezing step was associated with a high ratio of unsaturated to saturated fatty acids, a low C18:0 content, and high C16:0 and cyclic C19:0 relative concentrations. High resistance during frozen storage was related to a high cycC19:0 concentration. Finally, the low cryotolerance observed after fermentation at pH 4.5 was explained by a low C18:2 content.