Particulate membrane fractions from Volvox carteri catalyze the transfer of mannose from GDP-mannose to dolichyl diphosphate-[14C]chitobiose to form lipid-linked oligosaccharides up to a dolichyl diphosphate-chitobiose-(mannose)5 structure. Mannosylation of the chitobiosyl lipid requires divalent cations and detergents as solubilizing agents. Depending on the nature of the detergent, the oligosaccharide pattern differs markedly: With deoxycholate or the zwitterionic detergent 3-14 a lipid-linked trisaccharide accumulates. The nonionic Triton X-100, however, gives rise to a spectrum of compounds up to a heptasaccharide. Enzyme digestion of the tri- and pentasaccharide structure, obtained after mild acid hydrolysis of the corresponding [14C]glycolipids, revealed that the first mannose is bound via a beta-glycosidic linkage to the chitobiosyl core, whereas the outer mannose residues are linked as alpha-mannosides. Our studies indicate that, in agreement with recent findings in other organisms, the innermost alpha-mannosidic residues are donated directly from GDP-mannose. The structure of oligosaccharides synthesized by Volvox membranes is thus consistent with results from other eucaryotic species, suggesting a common pathway of N-glycosylation of glycoproteins.