The relative expression of large (L) and small (S) isoforms of the myelin-associated glycoprotein (MAG) and their glycosylation were compared in developing spinal cord of quaking and control mice. Using antisera specific for L- and S-MAG, respectively, it was shown that S-MAG is the principal isoform in quaking mice at all ages between 13 and 72 days, although L-MAG was just detectable by western blotting at the early ages. Both L- and S-MAG have higher apparent molecular weights in quaking mice than in controls. Experiments involving lectin binding and glycosidase treatment demonstrated that the higher molecular weight of MAG in the quaking mutant was due to a higher content of N-acetylneuraminic acid residues linked alpha 2-3 to galactose as well as to more branching of oligosaccharide moieties indicated by a higher content of subterminal galactose residues. The total sialic acid measured by HPAE-chromatography in purified quaking MAG was 40% higher than in control MAG. By contrast, quaking MAG contained less of the adhesion-related, HNK-1 carbohydrate epitope. Another difference was that a lower molecular weight form of MAG with predominantly high mannose oligosaccharides was prominent in young quaking mice, but not in controls. The abnormalities of MAG expression related to splicing of its mRNA and glycosylation may contribute to the myelin pathology in quaking mutants.