The glycophosphosphingolipids of Tritrichomonas foetus, an aerotolerant parasite of the urogenital tract of cattle, have been characterized by a combination of metabolic labeling, chromatography, and tandem mass spectrometry. The acidic glycolipid fraction of T. foetus obtained by DEAE Sephadex A-25 column chromatography was subfractionated by high performance thin layer chromatography and the component lipids were purified by high performance liquid chromatography. Two nonsaponifiable lipid fractions, designated TF1 and TF2, could be metabolically labeled with [3H]myoinositol and [32P]orthophosphate. [3H]Fucose and [14C]ethanolamine were preferentially incorporated into the TF1 fraction. TF1 was partially hydrolyzed by α-fucosidase. Both TF1 and TF2 contain ceramides, the most abundant having either sphinganine or sphingosine and a 16:0 N-acyl group. TF2 contains inositolphosphoceramides. TF1, on the other hand, contains three closely related components, in each of which fucose is linked to inositol diphosphate with one of the phosphates linked to the ceramide moiety and the other phosphate either free or linked to ethanolamine or N-acetylethanolamine. TF1 appears to be a novel class of glycophosphosphingolipid which shows some structural similarities to the glycosylphosphatidylinositol anchors of eukaryotic membrane proteins. © 1991.