Marburg virus was propagated in E6 cells, a cloned cell line of Vero cells, in the presence of [6-3H]glucosamine Radiolabelled viral glycoprotein was digested with trypsin, and oligosaccharides were liberated by sequential treatment with endo-β-N-acetylglucosaminidase H, peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F and O-glycosidase, by β-elimination, and by alkaline hydrolysis. After fractionation by HPLC and gel filtration, glycans were characterized chromatographically, by digestion with exoglycosidases and, in part, by methylation analysis and liquid secondary ion mass spectrometry. The oligosaccharide structures thus established include oligomannosidic and hybrid-type N-glycans, as well as neutral fucosylated bitri- and tetraantennary species, most of which carry an additional bisecting N-acetylglucosamine. In addition, high amounts of neutral mucin-type O-glycans with type-1 and type-2 core structures were detected. None of the glycans present in this viral glycoprotein carried sialic acid residues.