Undegraded mucus glycoprotein has been isolated in highly purified form from gastric secretion of cystic fibrosis patients. The purification procedure involved gel filtrations on Bio-Gel P-100 and Bio-Gel A-50 and lipid extractions with five mixtures of the organic solvents. The final preparation represented pure glycoprotein as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, cesium chloride density gradient centrifugation, and lipid analysis. Treatment of the pure and delipidated glycoprotein with methanolic KOH or hydroxylamine resulted in liberation of ester-bound fatty acids. Of the total released fatty acids, 95% were represented by hexadecanoate (36.5%), octadecanoate (48.7%), and octadecenoate (8.6%). The quantitative analysis established that, on the average, 12.2 nmol of fatty acids/mg of glycoprotein were released. The studies on cystic fibrotic glycoprotein susceptibility to proteolytic digestion indicated that fraction of glycoprotein which was resistant to pronase digestion contained on the average 33.1 nmol of fatty acids/mg of glycoprotein. After removal of the fatty acid residues from pronase-resistant glycoprotein, by treatment with hydroxylamine, the glycoprotein became susceptible to proteolytic digestion. Thus, in cystic fibrosis, the covalently bound fatty acids interfere with proteolytic degradation of mucus glycoprotein. Perhaps this is the major defect of cystic fibrosis glycoproteins and the cause of the obstruction of secretory glands and the accumulation of poorly soluble secretions.