The dominant Chinese hamster ovary cell glycosylation mutant, LEC18, was selected for resistance to pea lectin (Pisum sativum agglutinin (PSA)). Lectin binding studies show that LEC18 cells express altered cell surface carbohydrates with markedly reduced binding to 125I-PSA and increased binding to 125I-labeled Datura stramonium agglutinin (DSA) compared with parental cells. Desialylated [3H]Glc-labeled LEC18 cellular glycopeptides that did not bind to concanavalin A-Sepharose exhibited an increased proportion of species that were bound to DSA-agarose. Most of these glycopeptides bound to ricin-agarose and were unique to LEC18 cells. This fraction was purified from approximately 10(10) cells and shown by 1H NMR spectroscopy and methylation linkage analysis to contain novel N-linked structures. Digestion of these glycopeptides with mixtures of beta-D-galactosidases and N-acetyl-beta-D-glucosaminidases gave core glycopeptides that, in contrast to cores from parental cells, were mainly not bound to concanavalin A-Sepharose or to PSA-agarose. 1H NMR spectroscopy, matrix-assisted laser desorption ionization/time of flight mass spectrometry, electrospray mass spectrometry, and collision-activated dissociation mass spectrometry showed that the LEC18 core glycopeptides contained a new GlcNAc residue that substitutes the core GlcNAc residues. Methylation linkage analysis of the parent compound provided evidence that the GlcNAc is linked at O-6 to give the following novel, N-linked core structure. [formula: see text]