Listeria monocytogenes , a Gram-positive pathogenic food-borne bacterium, is notably resistant to chill and osmotic stress. Listeria is characterized by an unusual proportion of branched-chain fatty acid (>85%), primarily anteiso-15:0 and anteiso-17:0. Cells grown at lower temperatures contain significantly less anteiso-17:0 than those grown at higher temperature. Cold-shock studies were performed to determine the mechanisms by which Listeria perceives cold shock and changes its membrane composition. This study made three comparisons: (i) The lipids of L. monocytogenes cultured at 30°C were compared with those of cells grown at 6°C. Extraction of total lipids yielded 4‐4·5 and 6·5‐7 mg ml -1 cell mass (wet weight), respectively. Total lipids contained 3‐4% and 2‐2·5% lipid phosphorus, respectively, reflecting a higher content of neutral lipids in cells grown at 6°C. (ii) The fatty-acid compositions of different lipid classes (total lipids, the major phospholipid, and the glyco-containing phospholipid class) all responded by increasing the proportion of anteiso-15:0 fatty acids at low temperature. (iii) The fatty-acid composition of the neutral and polar lipids from the early stage of the temperature response (3 days at 5°C) and the effect of chloramphenicol or cerulenin were examined. The results indicated that anteiso-15:0 fatty acids were selectively increased in the newly synthesized neutral lipids, but the polar lipids did not show an apparent change in fatty-acid composition. These changes in membrane fatty-acid composition did not require new protein synthesis (the process was chloramphenicol insensitive) and the existing condensing enzyme activity was necessary to the fatty-acid response (the process was cerulenin sensitive). © 1998 Academic Press