A glycophosphoceramide concentrate prepared from tobacco leaves was shown to contain a mixture of related lipids (Kaul, K., and Lester, R. L. (1975) Plant Physiol. 55, 120-129; Kaul, K., and Lester, R. L. (1978) Biochemistry 17, 3569-3575) with the simplest and most abundant components having the structure GlcN(+/- Ac)(alpha 1 leads to 4)GlcUA(alpha 1 leads to 2)myoinositol-1-O-phosphorylceramide (Hsieh, T. C.-Y., Kaul, K., Laine, R. A., and Lester, R. L. (1978) Biochemistry 17, 3575-3579). To determine the structure of the more complex members of this series, a mixture of oligosaccharides was prepared from a carboxyl-reduced glycophosphoceramide concentrate by alkali-catalyzed hydrolysis and alkaline phosphatase treatment. A combination of reverse-phase high pressure liquid chromatography (Wells, G. B., and Lester, R. L. (1979) Anal. Biochem. 97, 184-190), normal-phase high pressure liquid chromatography, and thin layer chromatography were used to resolve several oligosaccharides as acetylated derivatives. Products of methylation analysis, CrO3 oxidation, and deacetylation-deamination were identified using chemical ionization mass spectrometry to give the following novel structures. A major tetrasaccharide was completely characterized as Gal(alpha 1 leads to 4)GlcNAc(alpha 1 leads to 4)GlcUA(alpha 1 leads to 2)myoinositol. An additional structure of a minor tetrasaccharide was partially characterized as GlcNAc(alpha 1 leads to 4)GlcUA(alpha 1 leads to ?)myoinositol(O leads from 1 alpha)Man. These are representatives of a class of acidic glycolipids from plants, possibly analogous to the acidic gangliosides found in animal cell membranes.