This study reports a simple and sensitive method for determining the absolute configuration of the glycerol moieties in glycoglycerolipids. The method is based on chiral phase high-performance liquid chromatography (HPLC) separations of enantiomeric di- and monoacylglycerols released from glycosyldi- and monoacylglycerols, respectively, by periodate oxidation followed by hydrazinolysis. The released di- and monoacylglycerols were chromatographed as their 3,5-dinitrophenylurethane (3,5-DNPU), and bis(3,5-DNPU) derivatives, respectively. The derivatives were separated on two chiral phases of opposite configuration, (R)-and (S)-1-(1-naphthyl)ethylamine polymers for diacylglycerols and N-(R)-1-(1-naphthyl)ethylaminocarbonyl-(S)-valine and N-(S)-1-(1-naphyl)ethylamino-carbonyl-(R)-valine for monoacylglycerols. Clear enantiomer separations, which permit the assignment of the glycerol configuration, were achieved for sn-1,2(2,3)-dicyl- and sn-1(3)-monoacylglycerols generated from linseed oil triacylglycerols by partial Grignard degradation on all the chiral stationary phases employed. Using the method, we have determined the glycerol configuration in the glycosyl-diacylglycerols (monogalactosyl-, digalactosyl-, and sulfquinovo-syldiacylglycerols) and glycosylmonoacylglycerols (monogalactosyl-, digalactosyl-, and sulfoquinovosylmonoacylglycerols) isolated from spinach leaves and the coralline red alga Corallina pilulifera. The results clearly showed that the glycerol moieties in all the glycoglycerolipids examined have S-configuration sn-1,2-diacyl- and sn-1-monoacylglycerols). The new method demonstrates that chiral phase HPLC provides unambiguous information on the configuration of the glycerol backbone in natural glycosyldi- and monoacylglycerols, and that the two-step liberation of the free acylglycerols does not compromise glycerol chirality.