Mild alkaline hydrolysis of the glycophosphosphingolipids of the protozoan Leptomonas samueli liberated several phosphoinositol-containing oligosaccharides (PI-oligosaccharides), which were purified by high performance anion exchange chromatography. The oligosaccharides in the resulting four fractions were characterized by methylation analysis, fast atom bombardment mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy. The oligosaccharides contain the core structure Man alpha (1-4)GlcN alpha (1-6)-myo-inositol-1-OPO3, and are substituted with 2 mol of 2-aminoethylphosphonate per mol of oligosaccharide. The nonreducing ends of the oligosaccharides were terminated by rhamnose branched neutral and acidic xylose-containing penta-, hexa-, hepta- and octasaccharides, of which the three most abundant were shown to have the structures: [formula: see text] More tentative structures are also proposed for three minor oligosaccharides.