Abstract Different methods for extracting mucin components from human gastric mucosa have been evaluated. An extensive fractionation study of a product solubilised by pronase digestion is described. By chromatography of the crude extract on Bio-Gel P-30, a neutral glycopolypeptide fraction ( DP-1 ) of molecular weight >300,000 was obtained which had a composition similar to that of blood-group substance and contained most of the original carbohydrate. A second fraction, which was a mixture of glycopeptides, peptides, and nucleic acid, was further separated into neutral and acidic components. Another preparation of the glycopolypeptide was examined in greater detail. There was no evidence that this glycopolypeptide contained sulphate, contradicting the chemical investigations of other workers, but in agreement with known histochemical results. Evidence is presented that it consists of a polypeptide core composed preponderantly of threonine, serine, proline, alanine, and glycine, in which approximately 75% of the threonine and serine residues were involved in O -glycosidic linkages to oligosaccharide chains of variable length, through 2-acetamido-2-deoxygalactose residues. Structural investigations by periodate-oxidation studies and by gel chromatography of alkaline degradation products indicated that all of the 2-acetamido-2-deoxygalactose residues were substituted at O-6 and more than 85% at O-3.