NOR is a rare inheritable polyagglutination phenomenon that has been described in two families. Our recent studies on these erythrocytes showed they contained at least two unique neutral glycosphingolipids, and based on their reactivity with Griffonia simplicifolia IB4 (GSL-IB4) isolectin (Kusnierz-Alejska, G., Duk, M., Storry, J. R., Reid, M. E., Wiecek, B., Seyfried, H., and Lisowska, E. (1999) Transfusion 39, 32-38), both oligosaccharide chains terminated with an alpha-galactose residue. The reactivity with GSL-IB4 suggested that these oligosaccharide chains terminated with a Galalpha1-->3Gal- sequence and that anti-NOR agglutinins were common human anti-Galalpha1-->3Gal xenoantibodies. In this report we describe the structure of one NOR component (NOR1) that migrated on thin-layer chromatographic plates in the region of pentaglycosylceramides. Treatment of this sample with alpha-galactosidase and beta-N-acetylhexosaminidase was followed by high-performance thin-layer chromatography with product detection by lectins and the anti-Gb4 monoclonal antibody. The results suggested that NOR1 was an alpha-galactosylated Gb4Cer with a beta-N-acetylhexosaminidase-resistant GalNAc residue. Gas phase disassembly by ion trap mass spectrometry analysis showed the sequence to be Hex1-->4HexN1-->3Hex1-->4Hex1-->4Hex linked to a ceramide composed of C18 sphingosine and a C24 monounsaturated fatty acid. Together these data indicate NOR1 to be a novel Galalpha1-->4GalNAcbeta1-->3Galalpha1-->4Galbeta1-->4 Glc-Cer structure. Additionally it has been shown that NOR glycolipids are recognized by human antibodies that were distinct from the known anti-Galalpha1-->3Gal xenoantibodies.