The flagellar pocket of the bloodstream form of the African sleeping sickness parasite Trypanosoma brucei contains material that binds the beta-D-galactose-specific lectin ricin (Brickman, M. J., and Balber, A. E. ( 1990) J. Protozool. 37, 219 - 224). Glycoproteins were solubilized from bloodstream form T. brucei cells in 8 M urea and 3% SDS and purified by ricin affinity chromatography. Essentially all binding of ricin to these glycoproteins was abrogated by treatment with peptide N-glycosidase, showing that the ricin ligands are attached to glycoproteins via N-glycosidic linkages to asparagine residues. Glycans released by peptide N-glycosidase were resolved by Bio-Gel P-4 gel filtration into two fractions: a low molecular mass mannose-rich fraction and a high molecular mass galactose and N-acetylglucosamine-rich fraction. The latter fraction was further separated by high pH anion exchange chromatography and analyzed by gas chromatography mass spectrometry, one- and two-dimensional NMR, electrospray mass spectrometry, and methylation linkage analysis. The high molecular mass ricin-binding N-glycans are based on a conventional Manalpha1-3( Manalpha1-6) Manbeta1-4GlcNAc beta1-4GlcNAc core structure and contain poly-N-acetyllactosamine chains. A significant proportion of these structures are extremely large and of unusual structure. They contain an average of 54 N-acetyllactosamine (Galbeta1-4GlcNAc) repeats per glycan, linked mostly by - 4GlcNAcbeta1-6Galbeta1- interrepeat linkages, with an average of one - 4Glc-NAcbeta1- 3(-4GlcNAcbeta1 -6) Galbeta1- branch point in every six repeats. These structures, which also bind tomato lectin, are twice the size reported for the largest mammalian poly-N- acetyllactosamine N-linked glycans and also differ in their preponderance of - 4GlcNAcbeta1 - 6Galbeta1- over - 4GlcNacbeta1-3Galbeta1-interrepeat linkages. Molecular modeling suggests that - 4GlcNAcbeta1 - 6Galbeta1- interrepeat linkages produce relatively compact structures that may give these giant N- linked glycans unique physicochemical properties. Fluorescence microscopy using fluorescein isothiocyanate-ricin indicates that ricin ligands are located mainly in the flagellar pocket and in the endosomal/lysosomal system of the trypanosome.