Glycosphingolipids were isolated from primary cultures of porcine endothelial cells labelled with 14C-galactose or 14C-glucosamine. They were characterized by their mobility on thin layer chromatogram, their sensitivity to exoglycosidases, and their labelling with antibodies. In addition to the major glycosphingolipids, globotetra- and globotriaosylceramide, minor ones were identified as penta- and heptaglycosylceramide of the neolactoseries terminated by either Gal alpha 1-3Gal- (xenoreactive epitope) or Fuc alpha 1-2Gal- (H determinant). Two gangliosides were found, GM3 and GD3, and N-glycolylneuraminic acid was their major sialic acid. Therefore, porcine endothelial cells differ from human endothelial cells by expression of glycosphingolipids that are absent in man: two Gal alpha 1-3Gal-terminated glycolipids recognized by human natural antibodies, and two N-glycolylneuraminic acid-terminated gangliosides which are potent immunogens.