A sulfotransferase activity which catalyzes the transfer of a sulfate group from 3'-phosphoadenosine-5'-phosphosulfate to gastric mucus glycoprotein has been demonstrated in the rat gastric mucosa. Subcellular fractionation studies revealed that the enzyme activity was present in a Golgi-rich membrane fraction. Optimum enzymatic activity for sulfation of mucus glycoprotein was obtained with 0.5% Triton X-100 and 30 mM NaF at a pH of 6.8. The apparent Km of the enzyme for gastric mucus glycoprotein was 10.5 microM, and the sulfate ester was found incorporated into the carbohydrate chains of mucin. The sulfo-transferase activity of the Golgi enzyme was stimulated by sofalcone, while reduction in the rate of sulfation occurred in the presence of sucralfate and aspirin. The rate of enhancement of mucin sulfation by sofalcone was proportional to the drug concentration up to 5 X 10(-7) M, at which concentration a 17% increase in the glycoprotein sulfation was attained. The rate of inhibition of mucin sulfation was proportional to concentrations of aspirin up to 3 X 10(-4) M and of sucralfate up to 1 X 10(-4) M, at which concentrations about 50% reduction in sulfotransferase activity was obtained. The apparent KI values calculated from the double-reciprocal plots were 15.1 microM for aspirin and 19.6 microM for sucralfate. The results suggest that although both sucralfate and sofalcone are potent gastric mucosal strengthening agents, only sofalcone is capable of enhancement of the sulfotransferase enzyme involved in gastric mucin sulfation.