A method was established for recovering the mucus gel layer of rat gastric mucosa without damage to underlying surface epithelium. The mucus gel was solubilized by stirring the gastric mucosa in a solution of N-acetylcysteine (NAC), a mucolytic agent. Optimal mucus gel solubilization was possible by treatment with 2% NAC for 5 minutes at room temperature. Mucus glycoprotein was quantitatively extracted and measured from the mucus gel sample obtained by the NAC treatment. This treatment caused no damage to surface epithelial cells, as observed by a light microscope. Besides NAC, pronase solution was also adequate for solubilizing the mucus gel layer without any damage to the surface epithelium. However, extraction and measurement of mucus glycoprotein from the pronase-treated mucus gel sample was not possible due to contamination by high molecular hexose-containing substances which were eluted along with the mucus glycoprotein from the column of Bio-Gel A-1.5m. This NAC method was used to examine changes in mucus glycoprotein content in the mucus gel at one hour following the oral administration of 16,16-dimethyl prostaglandin E2. A significant increase in mucus glycoprotein of the gel was brought about by the prostaglandin treatment. Thus, the present method was suitable for estimating the amount of mucus secreted in to the mucus gel layer.