The major unidentified polar lipid (compound X), recently demonstrated in the cell membrane of Mycoplasma fermentans, was purified by preparative silicic acid column chromatography. Chemical analyses of acid-hydrolyzed compound X revealed that, in addition to fatty acids, it contains glycerol, choline and phosphate in a molar ratio of approximately 1:1:2, and an amino acid that has a retention time similar to that of homoserine. The methylated fatty acid fraction of compound X was subjected to gas-liquid chromatography and revealed methyl palmitate and methyl stearate in a 4.6:1 molar ratio. The structure of compound X was further analyzed by combining mass spectrometry, 31P-NMR and 1H-NMR. The positive and negative fast atom bombardment spectra showed a major component of M(r) 1048 and a minor component of M(r) 1076. Two different phosphate groups were identified in each of the components by 31P-NMR. Fast atom bombardment, tandem mass spectrometry, negative and positive chemical ionization mass spectrometry together with mass spectra analyses of the water-soluble and ether-soluble products obtained by methanolysis has shown that, in addition to palmitic and stearic acid residues, the presence of glycerol, ribitol, cholinephosphate and homoserinephosphate residues. It is suggested that the apparent structure of compound X is either a phosphatidylcholine attached via a phosphotriester bond to a ribitolphosphohomoserine moiety or a phosphatidylhomoserine attached via a phosphotriester bond to a ribitolphosphocholine moiety. The major molecular species is the dipalmitoylderivative (M(r) 1048), whereas the minor molecular species is a stearoyl palmitoyl derivative (M(r) 1076).