The mechanism by which neomycin treatment reduces circulating ammonia concentrations was studied in normal and portacaval shunted rats. Rats were given neomycin for 3 days and then fasted for 24 hours to eliminate feces. Neomycin decreased arteriovenous differences of ammonia across the intestine even when the intestines were empty. Neomycin treatment lowered the activity of glutaminase in the intestinal mucosa and the rate of ammonia production from glutamine by isolated intestinal segments. The intestines from portacaval shunted rats had higher glutaminase activity (by 57%), and produced ammonia from glutamine at a greater rate (by 31%), than intestines from controls. Neomycin treatment lowered glutaminase activity and ammonia production in shunted rats, but glutaminase activity still remained higher than in controls (by 23%). The data indicate that the mechanism by which neomycin lower plasma ammonia is owing, at least in part, to a direct effect on the intestines. Specifically, neomycin causes a reduction in mucosal glutaminase activity and thereby decreases the ability of the mucosa to consume glutamine and produce ammonia.