Native (nonreduced) glutenin aggregates of two hard red spring wheat flours (Len of good quality and line 205 of poor quality) containing the same high molecular weight glutenin subunits (2*, 7+9, and 5+10) were investigated for the possibility of glycosylation. Glutenins isolated by pH-precipitation were separated and purified under nonreducing conditions into five different molecular weight species by multistacking SDS-PAGE and by transfer to polyvinylidene difluoride (PVDF) membranes by electroblotting. Carbohydrate compositions of the total glutenin fraction and of the different molecular weight glutenin species separated on the stacking gels (4–12% acrylamide) were determined. More total carbohydrates were found in the total glutenin of the line 205 flour (2.33%) than in that of the Len flour (1.87%), with glucose and xylose contributing to the greater total amount in line 205. However, the total glutenin of Len had approximately twice as much of the total of arabinose, mannose, and galactose as line 205 had. After purification by electrophoresis, smaller amounts of monosaccharides (glucose, xylose, arabinose, galactose, and mannose) in the different molecular species were detected. After electroblotting to PVDF membrane to increase purification, no arabinoxylans were found. Following extraction of glutenin from membranes, the β-elimination procedure in mild base under reducing conditions was used. In that procedure, the mono- or oligosaccharide side chains are released from the protein core, and the sugars originally involved in the protein-sugar linkage are reduced to the sugar alcohol. After derivatization (with trimethylsilyl), samples were analyzed by gas chromatography and mass spectrometry. Glucose, galactose, and a small amount of mannitol were found in most of the different glutenin aggregates, except that mannitol was not found in the 8% stacking gel glutenin fraction. The content of mannitol was greater in the higher molecular weight glutenin species at the 4 and 6% origins. These results support the hypothesis that the carbohydrates and protein in the glutenin macropolymer may be covalently linked.