The structural requirements for the interaction of the Asn-linked poly-N-acetyllactosamine-type oligosaccharide moieties of glycoproteins with various N-acetylglucosamine-binding lectins were investigated by means of affinity chromatography on immobilized lectin-Sepharose columns. High molecular weight glycopeptides containing poly-N-acetyllactosamine-type oligosaccharides obtained by Pronase digestion of human erythrocyte ghosts were treated with 0.1 M trifluoroacetic acid at 100 degrees C for 40 min and then several oligosaccharide fragments were purified with an amino-bonded silica column. Among these oligosaccharide fragments, trisaccharide Gal beta 1-4GlcNAc beta 1-6Galol bound to the wheat germ agglutinin (WGA)- and pokeweed mitogen (PWM)-Sepharose columns, and also showed affinity to the Datura stramonium agglutinin (DSA)-, Lycopersicon esculentum (tomato) agglutinin- and Solanum tuberosum (potato) agglutinin-Sepharose columns. Pentasaccharide Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Galol showed weaker affinity to the WGA- and PWM-Sepharose columns, compared to the trisaccharide. Trisaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Galol showed weak affinity to the WGA-Sepharose column and did not show any affinity to the other lectin-Sepharose columns. Hexasaccharide Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAcol bound only to the DSA-Sepharose column, indicating that only DSA does not require a GlcNAc beta(1-6)- linkage for interaction.