The mechanism of regulation of mucus production in the gastric mucosa remains unclear. Recently, we established a gastric surface mucous cell line GSM06, which produces periodic acid-Shiff (PAS)-positive glycoconjugate (mucus) layers on the cell surface, from transgenic mice harboring a temperature-sensitive simian virus 40 large T-antigen gene. In this study, GSM06 cells were examined for its production of PAS-positive glycoconjugate layers to acid secretagogues and growth factors. The cells were cultured at nonpermissive temperature (39°C) for 3-18 days and stained with PAS. Insulin (1-30 μg/ml; 0.29-8.6 μM) time- and dose-dependently increased production of glycoconjugates on the cell surface. When glycoconjugate layers produced by stimulation of insulin (3-30 μg/ml; 0.86-8.6 μM) were removed from the cell surface of GSM06 cells by a mild trypsin treatment, PAS-positive materials were remarkably decreased (day 18). In addition, morphological findings indicate that a high concentration of insulin (30 μg/ml; 8.6 μM) produced thick PAS-positive glycoconjugate layers just like normal gastric surface mucosa on the cell surface on day 18. In contrast, histamine (0.1-100 μM), carbachol (0.1-100 μM), gastrin-17 (0.1-100nM), epidermal growth factor (0.01-10 ng/ ml; 1.7-1,700 pM), transforming growth factor-α (0.01-10 ng/ml; 1.8-1,800 pM), and fetal bovine serum (1-10%) did not increase glycoconjugate production. These findings suggest that insulin is a stimulator of glycoconjugate production, and stimulates production of glycoconjugate layers on the cell surface in the gastric surface mucous cell l