Olfactomedin is the major glycoprotein of the extracellular mucous matrix of frog olfactory neuroepithelium. It is responsible for the primary architecture of this extracellular matrix by forming via intermolecular disulfide bonds polymers, which are covered with evenly spaced carbohydrate groups. To study glycosylation of olfactomedin, we raised antibodies against the mature protein and antibodies against a region adjacent to an N-linked glycosylation site near its amino terminus. The latter antibodies cannot bind when this site is glycosylated and reveal precursors of olfactomedin in the perinuclear regions of Bowman's glands. In contrast, antiserum against the mature protein stains acinar regions of glands and the ciliary surface. Enzymatic deglycosylation of olfactomedin shows stepwise removal of carbohydrate and reveals a 51-kDa deglycosylated form. Our results indicate that, prior to secretion, most, if not all, of the six potential N-linked glycosylation sites of olfactomedin are glycosylated with carbohydrate moieties of about 8-10 sugar residues.