The effect of ethanol on the synthesis and secretion of mucus glycoprotein in gastric mucosal cells was investigated. The mucosal cell suspensions were subjected to a short-term (4 h) culture in the presence of 0-1.5 M ethanol, with [3H]proline and [3H]palmitic acid as markers for glycoprotein synthesis and acylation. The synthesized labeled mucus glycoprotein was isolated from the incubation medium (extracellular glycoprotein) and from the mucosal cells (intracellular glycoprotein), and analyzed. Depending upon the ethanol concentration in the cell culture medium, two distinct effects on the synthesis and secretion of mucus glycoprotein were observed. The cells cultured in the presence of 0.02-0.1 M ethanol showed increased ability for the incorporation of [3H]proline and [3H]palmitic acid, and for the secretion of the newly assembled mucus glycoprotein. The synthesis of the glycoprotein increased 18-fold, acylation 5-fold, and secretion 10-fold. The synthesized glycoprotein, however, contained four to five times less of acyl-bound fatty acids. Ethanol at 0.1-1.5 M caused a marked reduction (62-64%) in the mucus glycoprotein synthesis, but the amount of glycoprotein released to the medium remained constant. This indicated that higher concentrations of ethanol caused the release of the preformed intracellular mucus glycoprotein reserves. The results demonstrate that gastric mucosal cells incubated in the presence of ethanol exhibit impaired synthesis and secretion of mucus glycoprotein, and that the severity of impairment depends upon the ethanol concentration.