A mixture of two lyso isomers of a galactolipid was obtained from Dictyonema glabratum. Aqueous hydrolysis gave rise to galactose and glycerol in a 3:1 molar ratio. ESI-MS spectroscopy gave, in the positive-ion mode, a pseudomolecular ion at m/z 839 and daughter ions with m/z 677, 600, 515 and 353, suggesting three galactosyl units linked to a glycerol moiety, substituted by one O-acyl group. 1D and 2D NMR experiments were used to characterize the glycolipid, and HMQC examination showed three anomeric signals, corresponding to two alpha-Galp and one beta-Galp residue liked to glycerol. The glycolipid structure was shown to be O-alpha-D-Galp-(1-->6)-O-alpha-D-Galp-(1-->6)-O-beta-D-Galp-(1<-->1)-2- and -3-monoacyl-D-glycerol, the latter structures not having been previously found in nature. The fatty acid composition was determined by GC-MS of derived methyl esters: that of palmitic acid C(16:0) was the most abundant, although the presence of C(12:0), C(14:0), C(16:1) and C(18:0) esters was observed.