The structure of one glucolipid from the membrane of Acholeplasma laidlawii, strain A-EF22, was determined. This glucolipid is synthesized only when a large fraction of saturated, straight-chain fatty acids are incorporated into the membrane lipids of strain A-EF22. The lipid was studied by 1H- and 13C-NMR spectroscopy. The structure of the lipid is 1,2-diacyl-3-O-[6-O-acyl-(alpha-D-glucopyranosyl)]-sn-glycerol. The result for this lipid shows that a previously published structure, based on incomplete chemical analyses, was incorrect. The phase equilibria for 1,2-diacyl-3-O-[6-O-acyl-(alpha-D-glucopyranosyl)]- sn-glycerol and the two dominating lipids in A. laidlawii, monoglucosyldiacylglycerol and diglucosyldiacylglycerol, are discussed and related to the chemical structure of the lipids.