Thin layer chromatograms of ostrich liver neutral glycosphingolipids were immunostained with human sera. In addition to the expected staining of the Forssman pentaglycosylceramide by some sera, more polar and less abundant unknown glycolipids could be stained. Among them, the shortest carbohydrate chain glycolipid was purified and structurally characterized by mass spectrometry, proton NMR and methylation analysis. It was a novel pentaglycosylceramide of the neolactoseries terminated with the Gal(beta1-4)Gal determinant which is not expressed in mammalian species. Human antibodies affinity-purified on a synthetic Gal(beta1-4)Gal(beta1-4)Glc-Sepharose column recognized the newly characterized Gal(beta1-4)Gal-terminated pentaglycosylceramide, and, in addition, longer chain glycolipids. Occurrence of antibodies directed at the Gal(beta1-4)Gal epitope was studied by ELISA on 108 human sera. Anti-Gal(beta1-4)Gal antibodies were predominantly IgM, and their distribution was similar to that of anti-Gal(alpha1-3)Gal and anti-Forssman IgMs. It was concluded that anti-Gal(beta1-4)Gal are natural antibodies, not previously identified in man. They can be considered as xenoantibodies directed at species which express Gal(beta1-4)Gal-terminated carbohydrate chains.