Defining glycosphingolipid structures in species amenable to genetic manipulation, such as Drosophila melanogaster, provides a foundation for investigating mechanisms that regulate glycolipid expression. Therefore, eight of the 12 major glycosphingolipids, accounting for 64% of lipid-linked carbohydrate in Drosophila embryos, were purified after separation into acidic and zwitterionic pools. The zwitterionic lipids possess phosphoethanolamine (PEtn) linked to one or more GlcNAc residues and comprise a family of serially related structures. The longest characterized glycolipid, an octaosylceramide, designated Nz28, has the structure: GalNAcbeta, 4(PEtn-6)GlcNAcbeta,3Galbeta,3GalNAcalpha,4Ga lNAcbeta, 4(PEtn-6)GlcNAcbeta,3Manbeta,4GlcbetaCer. Heptaosyl (Nz7), hexaosyl (Nz6), pentaosyl (Nz5) and tetraosyl (Nz4) forms of Nz28, sequentially truncated from the nonreducing terminus, possess only one PEtn moiety. The major acidic lipid, designated Az29, possesses two PEtn moieties and a glucuronic acid linked to a Gal-extended Nz28. Two other acidic glycolipids, Az9 and Az6, exhibit one PEtn moiety and the same hexose and N-acetylhexosamine composition as Az29 and Nz6, respectively. The fully extended Drosophila core oligosaccharide differs from that of other dipterans in the linkage at a single glycosidic bond, a distinction with significant structural and biosynthetic consequences. Furthermore, acidic species account for a larger proportion of total glycosphingolipid, and PEtn substitution of GlcNAc is more complete in the Drosophila embryo. Divergent characteristics may reflect interspecies variation or stage-specific glycosphingolipid expression in dipterans.