A glycoprotein was isolated from rat-colonic mucosa. Analytical ultracentrifugation studies showed the glycoprotein to be homogeneous, having an apparent molecular weight of 9.0 X 10(5); no subunits could be detected in the presence of sodium dodecyl sulfate. It contained 14% of protein and 86% of carbohydrate. The principal sugars in the glycoprotein were galactose, fucose, sialic acid, 2-acetamido-2-deoxygalactose, and 2-acetamido-2-deoxyglucose. A small proportion of mannose was also present. The glycoprotein, apart from the usual carbohydrate constituents present in mucus glycoproteins, contained sulfate, but no uronic acid. High amounts of serine and threonine, and low contents of aromatic and traces of sulfur-containing amino acids, reflect a similarity of this glycoprotein to other mammalian mucus glycoproteins; it differs, however, by its high proportions of Asx + Glx (26 mol.%). Cleavage studies with alkaline borohydride indicated O-glycosidic linkages between N-acetylhexosamine and serine, and threonine, of the peptide core in the glycoprotein. Only about one third of the serine and threonine was linked to the carbohydrate side-chains, which averaged about 22 units in length and were apparently branched.