The lipid composition of Treponema pallidum Kazan 5 cultivated in a lipid-defined medium was investigated. Lipids comprised 18 to 20% of the dry weight of the treponeme. Glycolipid and phospholipids accounted for 90 to 95% of the total lipids and free fatty acids made up the remaining 5 to 10%. The major polar lipids were the glycolipid, 1-(O-α-d-galactopyranosyl)-2,3-diglyceride (45 to 55%), and phosphatidylcholine (30 to 40%). Phosphatidylethanolamine (5 to 10%), an unidentified compound (1 to 2%), and occasional trace amounts of diphosphatidylglycerol (cardiolipin) were also found. The monogalactosyldiglyceride was also a major component (50%) of the lipids of the Reiter, Noguchi, and Nichols strains of T. pallidum. The fatty acid composition of Kazan 5 usually consisted of saturated and unsaturated fatty acids ranging from 14 to 18 carbons depending upon the fatty acids added to the culture medium. When the cells were cultivated on elaidic acid (trans-9-octadecenoic acid), their lipids contained only elaidic acid.