Tumor-necrosis factor-α, produced by human B-cell lymphoblastoid cell line BALL-1, was expressed as four protein bands on SDS/PAGE analysis. It may have been glycosylated, based on the fact that the heavier two of the four bands disappeared after neuraminidase treatment. Sugar composition analyses revealed that the tumor necrosis factor-α contained galactose, N-acetylgalactosamine and N-acetylneuraminic acid as sugar components. To prepare sugar chains, tumor necrosis factor-α was treated with alkaline borodeuteride and the oligosaccharide-alditols liberated were fractionated by gel-filtration chromatography on a Bio-Gel P-4 column, followed by normal-phase HPLC. Three oligosaccharide-alditols were obtained, and the structures of two of them were identified by methylation analysis and exoglycosidase digestion. The structures of these oligosaccharide-alditols were Galβ1–3(NeuAcα2–6)GalNAcol and Galβ1–3GalNcol (GalNAcol, N-acetylgalactosaminitol). The structure of the remaining oligosaccharide-alditol was determined to be NeuAcα2–3Gal1–3GalNAcol by composition and methylation analyses. About 20% of tumor necrosis factor-α was found to be O-glycosylated, based on the results of the sugar composition and structure analyses. An amino acid sequence analysis of the glycosylated peptides was performed after Staphylococcus aureus V8 protease digestion of tumor necrosis factor-α had been completed, and it was proved that the O-glycosylation site of tumor necrosis factor-α was Ser 4.