The gastric H,K-ATPase is responsible for acid secretion by parietal cells. Its β-subunit is a glycoprotein which is exposed to the harsh, acidic environment of the stomach. The location and structural features of the N- linked oligosaccharides were determined using matrix-assisted laser desorption ionization mass spectrometry (MALDI/MS) (in conjunction with mass' composition analysis and exoglycosidase digestions), Edman degradation, and monosaccharide composition analysis. All seven N-linked sequons at positions 99, 103, 130, 146, 161,193, and 222 were fully glycosylated. An unusual restricted array of oligosaccharides was observed at individual Asn residues. Asn99 was modified exclusively with oligomannosidic-type structures (Man6GlcNAc2-Man8GlcNAc2). Asn193 contained both oligomannosidic (Man5GlcNAc2-Man8GlcNAc2) and lactosamine-type structures, indicating significant 'leakiness' in the pathway which converts oligomannose to lactosamine-type at a single glycosylation site. MALDI/MS with collision- induced dissociation was required to demonstrate that sequons separated by a single residue (99Asn-Ile-Ser-Asp-Asn-Arg-Thr105) were modified with only oligomannose and lactosamine structures, respectively. Analysis of the total oligosaccharide pool using MALDI/MS and exoglycosidase analysis revealed 24 lactosamine species (bi-, tri-, and tetraantennary structures), with all branches terminated in α-linked Gal residues, most possessing a single Fuc residue. Nine novel oligosaccharides contained multiple α-linked Gal residues per branch. Bi- and triantennary structures, with and without lactosamine repeats, were observed at Asn146 and Ash161. Tetraantennary structures with lactosamine repeats were found only at Asn130, and this site also contained most of the structures with multiple α-linked Gal residues per branch.