Ammonia production by eight groups of intestinal bacteria was measured, and the effect on ammonia production of lowered pH and ambient ammonia concentration was determined. Endogenous ammonia production from bacterial protoplasm was also examined. To examine the mechanisms by which fermentable substrates reduce ammonia formation in a faecal incubation system, the effect of lactose, lactulose or glucose on ammonia release by pure cultures of intestinal bacteria was studied. The largest amounts of ammonia were generated by gram-negative anaerobes, clostridia, enterobacteria, and Bacillus spp. Gram-positive non-sporing anaerobes, streptococci and micrococci formed modest amounts, and lactobacilli and yeasts formed very little ammonia. All groups of bacteria formed less ammonia at pH 5.0 than at pH 7.0 and production of ammonia was not inhibited when 30 mmol ammonia/litre was included in the medium. Small amounts of ammonia were formed due to endogenous metabolism of bacterial cells. Washed cell suspensions of four isolates of Bacteroides, one clostridial isolate and two streptococcal isolates formed less ammonia from alanine, methionine or histidine after growth in the presence of either lactose or lactulose. In contrast, the Bacteroides isolates formed more ammonia from aspartate than from either lactose or lactulose. Also, cultures of gram-negative anaerobes and enterobacteria, and to a lesser extent clostridia and streptococci, formed significantly less ammonia in nutrient broth when lactose, lactulose or glucose was included in the medium. This decrease in ammonia formation was not due to a fall in pH of the medium. Ammonia production by gram-positive non-sporing anaerobes was not affected by carbohydrate fermentation. These results suggest that gram-negative anaerobic bacteria make a major contribution to ammonia generated from peptides and amino acids in vivo, and that ammonia may be formed from bacterial cells in the colon. Fermentation of lactose and lactulose may repress the formation and inhibit the activity of enzymes responsible for ammonia release. In the human colon these substrate effects may decrease the amount of ammonia available to exert a toxic effect on the host, and thus contribute to the beneficial effects of lactulose when it is used in the treatment of portosystemic encephalopathy.