Phospholipids of many cold-tolerant organisms have been reported to contain more unsaturated fatty acids than cold-susceptible organisms, a phenomenon known to maintain membrane fluidity at low temperature. However, we have obtained results to the contrary through a comparison of the membrane phospholipids of six temperate and subtropical species belonging to the Drosophila melanogaster species group. With enhancement of cold tolerance, the percentages of monoenoic acids increased but the percentages of dienoic acids decreased, that is, the number of double bonds in the phospholipid decreased without a marked variation in the percentages of unsaturated fatty acids. Concomitantly, the percentage of fatty acids containing 16 carbon atoms increased, while that of fatty acids with 18 carbon atoms decreased. Since phosphatidylethanolamine is a dominant phospholipid in Drosophila, these changes probably contribute to keeping the homeoviscosity of the cellular membranes in a manner different to that in phosphatidylcholine-rich membranes, thereby increasing cold tolerance.