The novel amphoteric glycosphingolipids containing a choline phosphate were purified from whole tissues of the earthworm, Pheretima hilgendorfi. Their chemical structures were completely characterized as cholinephosphoryl-->6(Man alpha 1-4)Gal beta 1-6Gal beta 1-1Cer (cholinephosphorylmannosylneogalabiaosylceramide, named PGL3a) and cholinephosphoryl-->6Gal beta 1-6Gal beta 1-6Gal beta 1-1Cer (cholinephosphorylneogalatriaosylceramide, named PGL3b) by compositional sugar, fatty acid and sphingoid analyses, hydrogen fluoride degradation, partial acid hydrolysis, methylation analysis, exoglycosidase degradation, proton magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. The ceramide moieties of these lipids consisted of 22:0, 23:0 and 24:0 acids as major fatty acids, and branched octadeca- and nonadeca-4-sphingenines and octadeca-4-sphingenine as main sphingoids. Since the oligosaccharides and the ceramide moieties of PGL3a and PGL3b were identical with those of neutral glycosphingolipids found in this organism, the biosynthesis of the amphoteric glycolipids may occur by the addition of a choline phosphate residue to the corresponding neutral glycolipids, Man alpha 1-4Gal beta 1-6Gal beta 1-1Cer or Gal beta 1-6Gal beta 1-6Gal beta 1-1Cer.