Recombinant human granulocyte colony stimulating factor (G-CSF) produced in Chinese hamster ovary cells is glycosylated. The carbohydrate compositional analysis indicated that G-CSF molecule contains sialic acid, galactose and galactosamine. By isolation and characterization of the purified glycopeptides obtained from cleavages by Staphylococcal aureus V-8 protease and cyanogen bromide, the O-linked glycosylation site was confirmed to be a Thr residue at position 133. Neuraminidase and O-glycanase digestion followed by sodium dodecyl sulfate polyacrylamide and isoelectric focusing gel electrophoreses distinguished two possible carbohydrate structures attached at Thr-133: structure A, NeuNAc-Gal-beta(1,3)-GalNAc-O-Thr; and structure B, NeuNAc-Gal-beta(1,3)-[NeuNAc]-GalNAc-O-Thr. Different glycoforms, undigested or after glycosidase digestion, can also be separated by ion-exchange or reversed-phase high-performance liquid chromatography. The approach described in this report provides a simple and valuable procedure to characterize glycoprotein structures containing simple carbohydrate moieties.