The present work is directed to distinguish between ammonia production by the mucosa and by the intestinal flora, as well as to evaluate the influence of neomycin and lactulose. In vitro studies using rat intestine show that mucosa cells produce ammonia alanine and glutamic acid when incubated with glutamine, whose process can be impaired by neomycin or lactulose. Since the release of the above solutes is virtually the same in germ-free rats, the influence of the bacterial flora might be negligible under the experimental conditions used. Elimination of the aerobic microorganisms results in a minute decrease of ammonia concentration in portal blood in contrast to elimination of the anaerobic flora, which leads to an excessive reduction of ammonia formation. In germ-free rats colonisation with anaerobic microorganisms results in an increment in portal ammonia concentration, whose value, however, is still below levels observed in normal animals. Colonisation with aerobic bacteria has no effect on portal ammonia concentration. Neomycin and lactulose affect ammonia production in the gut by interfering with glutamine uptake in the mucosa cell, thus the influence upon ammonia formation apparently can not be exclusively explained by alterations of the intestinal flora. Possible reasons for the considerable increase in arterial glutamine levels in normal rats are discussed.