Mycoplasma pneumoniae lipids were fractionated by combined silicic acid and diethylaminoethyl-cellulose chromatography. The lipid fractions were tested for complement-fixing activity and for blocking of metabolism-inhibiting antibodies in human and rabbit antisera to M. pneumoniae. Thin-layer chromatography and carbohydrate analysis of hydrolysates of the lipid fractions indicated that glycolipids were responsible for the specific serological activities of the lipid extracts of the organism. Phosphatidylglycerol, which accompanied the glycolipids, was serologically inactive when isolated free from glycolipids. Phosphatidylglycerol considerably enhanced the serological activity of the glycolipids, serving as an auxiliary lipid. Because of this auxiliary effect, minute quantities of contaminating glycolipids sufficed to render serological activity to "purified" phosphatidylgylcerol preparations, providing an explanation to the previous identification of the active lipid as a phospholipid.