Acholeplasma axanthum is one of the few procaryotes, and the only member of the Mollicutes, known to contain phosphosphingolipids. Examination of strain S743 for glycolipids revealed the presence of glucosides of cholesterol and galactosides of glycerol as the predominant glycolipids. The major component is acylated diglucosylcholesterol, followed by monogalactosyldiacylglycerol and monoglucosylcholesterol. The glucose residues of the sterol-based compounds appear to be alpha-linked pyranoses, while the galactose of the glycerol-based lipid is an alpha-linked furanose. The "glycolipid' fraction also contained N-(3-hydroxy)acyl sphinganines with varying degrees of O-acylation. None of these ceramide derivatives was linked to carbohydrate. The major glycolipid, tentatively identified as alpha-D-glucopyranosyl-(1 leads to 3)-(O-acyl)-alpha-D-glucopyranosyl-(1 leads to 3)-cholesterol, along with its deacylated derivative, appears to be the first reported instance of steryl diglycosides among procaryotes, in contrast to the steryl monoglycosides, which are common to other mycoplasmata and some spirochetes.