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1. Production of hydrogen ions by the rat proximal jejunum was investigated using the everted sac technique. 2. Acidification occurred in the absence of glucose, increasing on addition of glucose to reach a maximal value. An apparent Km of 1-78 mM was derived for the glucose-dependent process. 3. Acidification in the presence of glucose was inhibited by 10 mM-2:4-dinitrophenol, 10 mM phlorrhizin, 10 mM aminophylline and anaerobiosis. 4. Histamine, ethylenediamine tetraacetic acid (EDTA), ouabain and acetazolamide, compounds known to alter acid production in gastric mucosa had no effect on jejunal acidification. 5. Galactose and 3-O-methylglucose failed to increase acidification; in contrast, mannose and fructose did lead to increases, indicating metabolic origin of the hydrogen ions. 6. Serosal and mucosal lactate production were measured and the calculated percentage of hydrogen ions possibly derived from this source was shown to account for only a small proportion of acidification. 7. The greatest increase in acidification with minimal simultaneous production of lactate occurred with ATP which was shown not to enter intestinal tissues. 8. A hypothesis for acidification, that of the break-down at the mucosal surface of ATP from intracellular metabolic sources, is proposed and its relevance to the postulated microclimate is discussed.