The unicellular stercorarian protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas' disease. The epimastigote form of the parasite is covered in a dense coat of glycoinositol phospholipids and short glycosylphosphatidylinositol (GPI)-anchored mucin-like molecules. Here, we describe the purification and structural characterization of NETNES, a relatively minor but unusually complex glycoprotein that coexists with these major surface components. The mature glycoprotein is only 13 amino acids in length, with the sequence AQENETNESGSID, and exists in two forms with either four or five post-translational modifications. These are either one or two asparagine-linked oligo-mannose glycans, two linear alpha-mannose glycans linked to serine residues via phosphodiester linkages, and a GPI membrane anchor attached to the C-terminal aspartic acid residue. The variety and density of post-translational modifications on an unusually small peptide core make NETNES a unique type of glycoprotein. The N-glycans are predominantly Man alpha 1-6(Man alpha 1-3) Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc beta 1-Asn; the phosphate-linked glycans are a mixture of (Man alpha 1-2) (0-3)Man1-P-Ser; and the GPI anchor has the structure Man alpha 1-2(ethanolamine phosphate)Man alpha 1-2Man alpha 1-6Man alpha 1-4(2-aminoethylphosphonate-6)GlcN alpha 1-6-myo-inositol-1-P-3(sn-1-O-(C-16:0)alkyl-2-O-(C-16:0) acylglycerol). Four putative NETNES genes were found in the T. cruzi genome data base. These genes are predicted to encode 65-amino acid proteins with cleavable 26-amino acid N-terminal signal peptides and 26-amino acid C-terminal GPI addition signal peptides.