A macroglycolipid containing galactose and N-acetylglucosamine as predominant sugar constituents was prepared together with glycophorin from rabbit erythrocyte membranes by extraction with lithium diiodosalicylate and partition in aqueous phenol. The macroglycolipid was effectively separated from the glycophorin fraction by ion-exchange chromatography in the presence of a detergent, Ammonyx LO. Its yield (ca. 4 mg/100 ml erythrocytes) was significantly higher than that of the macroglycolipids from human erythrocytes. The structure of the carbohydrate moiety in the macroglycolipid was analyzed by methylation analysis, Smith degradation, nitrous acid deamination, and chromium trioxide oxidation. Assuming one ceramide residue per molecule, the average number of sugars in the macroglycolipid was about 30. The macroglycolipid had a highly branching structure: Gal(alpha 1 leads to 3)Gal(beta 1 leads to 4)GlcNAc sequences are present at nonreducing termini and leads to 3 Gal(beta 1-4)GlcNAc repeating units are present in the inner core of the sugar chain. Some of the inner galactose residues branch at the C-6 position. Constituents of the ceramide moiety were also characterized.