SAG1 (P30) is the major surface protein of the Toxoplasma gondii tachyzoite, the life cycle stage associated with the acute phase of infection. The protein is inserted into the parasite's plasma membrane by a glycosyl-phosphatidylinositol anchor, a modification that is present on all T. gondii surface proteins characterized so far. Here we describe a detailed structural analysis of this anchor. GPI anchor peptides were isolated from [3H]glucosamine labeled purified P30 by protease digestion and phase partitioning. Neutral glycans were prepared from this material by dephosphorylation and deamination. Two glycoforms were characterized by gel filtration and high performance ion exchange chromatography in combination with exoglycosidase treatment. Both forms were shown to carry an N-acetylgalactosamine bound to the first mannose of the conserved three-mannosyl core. Glycan B carries an additional terminal hexose linked to GalNAc. To identify the nature of this hexose, bulk anchor peptide was prepared and glycans were purified by aminopropyl-HPLC. Highly purified glycans were subjected to MALDI-TOF-MS and, after derivatization, to FAB-MS and methylation linkage analysis. The structures of the two anchors found on SAG1 were determined to be: Man-alpha1,2-Man-alpha1,6-Man-[GalNAc-beta1,4-]-alpha1,4-GlcN-PI and Man-alpha1,2-Man-alpha1,6-Man [Glc-alpha1,4-GalNAc-beta1,4-]-alpha1,4-GlcN-PI. Comparison of these structures with free GPI glycolipid precursors characterized in T. gondii suggests that core modification of the anchor takes place prior to transfer to the protein.