Affinity-purified glycopeptides were prepared from Trypanosoma cruzi using the carbohydrate-specific monoclonal antibody WIC29.26, These glycopeptides contain rhamnose, fucose, xylose, and galactose, in the ratio 1:1:2:3, A series of oligosaccharides was released from the glycopeptides by mild acid hydrolysis, while, in contrast, no oligosaccharides were released by either peptide N-glycosidase F or conventional base-catalyzed beta-elimination and reduction, This suggested the presence of a phosphodiester linkage between the carbohydrate and peptide, which was further supported by the detection of phosphothreonine in the glycopeptides, The mild acid liberated (MAL) fraction was resolved into two major acidic oligosaccharides (MAL-P1 and MAL-P2), two minor neutral oligosaccharides (MAL-P1b and MAL-P2b) and a neutral fraction (MAL-N1), consisting of Gal and Xyl monosaccharides, The MAL-P1 and MAL-P2 oligosaccharides proved to be hexa- and heptasaccharides that shared a common xylose reducing terminus, but differed by one galactofuranose residue, and their negative charge was shown to be due to the presence of cyclic-phosphate attached to nonreducing terminal galactofuranose residues, The MAL-P1b and MAL-P2b oligosaccharides appeared to be nonphosphorylated versions of MAL-P1 and MAL-P2. Partial structures of MAL-P1 and MAL-P2 are suggested, based on compositional analyses, electrospray mass spectrometry, and tandem mass spectrometry before and after permethylation. The origin and significance of these unique trypanosomatid glycoconjugates is discussed.