Using [³H]-labeled oligosaccharides, we found that the presence of at least two α-mannosyl residues with free hydroxyl groups at C-3, 4, and 6 is required for oligsaccharides to be retained by a concanavalin A-Sepharose column. This finding is also applicable to N-[^I4C]acetylated glycopeptides. Thus, the concanavalin A-Sepharose column might become a useful tool for structural studies of glycopeptides and oligosaccharides and for their fractionation. Glycopeptides prepared from the trypsinate of rat fibroblasts, which had been purified by paper electrophoresis, were further separated into two fractions by chromatoeraphy on a concanavalin A-Sepharose column.