The resistance to freezing and frozen storage of Streptococcus thermophilus was related to the fatty acid composition of the cell membrane. The effects of four experimental factors were investigated on the fatty acid concentrations and on the recovery of acidification activity of S. thermophilus stored at −20°C by using a complete experimental design: incorporating oleic acid in the culture medium, fermentation pH, addition of glycerol as cryoprotective agent and duration of storage. The acidi- fication activity decreased during the freezing and the frozen storage of S. thermophilus. The storage time slightly enhanced the unsaturated fatty acid concentrations. The addition of glycerol did not modify the fatty acid composition but increased the resistance to frozen storage. The addition of oleic acid and the decrease of the fermentation pH enhanced the ratio unsaturated:saturated fatty acids and improved the recovery of the acidification activity. These results indicate that the resistance to frozen storage was closely related to the membrane fatty acid composition. We interpreted this as an adaptation of S. thermophilus to the addition of oleic acid and the unfavorable growth conditions that corresponded to a low fermentation pH.