Unlike the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe synthesizes large outer chains on the N-linked oligosaccharides that consist mainly of D-Gal and D-Man residues. The fission yeast och1(+) gene product has alpha1,6-mannosyltransferase activity, and Och1p is the key enzyme in the initiation of outer chain elongation. Although the in vitro substrate specificity of S. pombe Och1p has been reported (Yoko-o et al., FEBS Lett., 489, 75-80 (2001)), the structure of the N-linked oligosaccharides of och1Delta cells has not been investigated. In this study, we report a structural analysis of S. pombe N-linked oligosaccharides. Lectin blot analysis indicated that galactose residues were attached to the cell surface glycoproteins of the och1Delta cells. We conducted a structural analysis of pyridylaminated N-linked oligosaccharides prepared from galactomannoproteins by HPLC and (1)H NMR. These analyses revealed that the N-linked oligosaccharides of the och1Delta cells displayed heterogeneity in the glycan consisting of Hex(11-15)GlcNAc(2). The structural heterogeneity arose mainly from the addition of alpha1,2- and alpha1,3-Gal residues to the Man(9)GlcNAc(2) core structure.