An in vitro fecal incubation system was used to demonstrate how lactulose influences ammonia metabolism in the colon. Lactulose and other fermentable substrates (glucose, mannitol, and sorbitol), pH and organic acid were varied independently so that their different effects could be determined. Fermentable substrate caused a fall in ammonia concentration during the period of fermentation. Acidification to pH 5.0 or less, with hydrochloric acid or a lactic-acetic acid mixture, significantly reduced ammonia generation, but unlike fermentable substrates, did not lower the existing ammonia concentration. The lactic-acetic acid mixture did not reduce ammonia generation significantly below that found with acidification by hydrochloric acid. The effect of lactulose in reducing ammonia concentration is attributed to its role as a bacterial substrate in either increasing bacterial assimilation of ammonia or reducing deamination of nitrogenous compounds. The effect of low pH in reducing generation of ammonia appears to be part of a general reduction in bacterial metabolism.