The NH2-terminal sialoglycopeptides from human erythrocyte glycophorin A have been obtained by specific proteolytic cleavage and gel filtration chromatography. By cyanogen bromide cleavage, a glycosylated octapeptide was obtained from blood group M donors having an amino acid composition and 13C NMR spectrum consistent with the structure (formula: see text) was demonstrated. By Staphylococcus aureus protease cleavage, a glycosylated pentapeptide was obtained from N donors having the same structure as II, without the carboxyl-terminal sequence Val . Ala . Hse. Methanolysis/gas chromatographic analysis and 13C NMR spectroscopy of I and II and their asialo derivatives reveal that the M- and N-active sialoglyco-octapeptides both have identical oligosaccharide structures, each containing three O-linked tetrasaccharides with the structure NeuNAc alpha 2-3Gal beta 1-3(NeuNAc alpha 2-6)GalNAc alpha 1-O-Ser(Thr). The demonstration of the anomeric form of GalNAc-peptide linkages revealed by 13C NMR has previously been inaccessible by chemical analysis. Conformationally, I and II appear identical and both manifest several unusual resonance shifts suggestive of a glycopeptide secondary structure involving four specific hydrogen bonds. Calcium ion titration was also found to induce shifts in the NeuNAc 13C resonances that may be of functional significance. Serological studies reveal that both the M and N glyco-octapeptides and the N glyco-pentapeptide retain all of the M and N activity of the parent structure. Deamination and/or desialylation completely destroys this activity. These data are consistent with a model in which the M or N determinant is the NH2-terminal amino acid and a NeuNAc residue(s). From these data it is concluded that there is no chemical basis for assertions in the literature that M and N antigens differ in their oligosaccharide structure or that the N antigen is biosynthetically transformed to the M antigen by sialylation.