Recent studies from this laboratory have determined that colonic K + absorption is altered by the PCO 2 and by secondary hyperaldosteronism. Partial inhibition by vanadate and mucosal ouabain suggested the operation of an H +/K + exchange pump. To determine the mechanism of acidification in rat distal colon, we measured in vitro acidification using the pH-stat technique by voltage-clamped segments of colonic epithelium in controls and in the presence of secondary hyperaldosteronism, induced by a sodium-deficient diet. Chronic stimulation with aldosterone resulted in increased mucosal acidification in vitro for at least 2 h. This effect could not be accounted for by lactate production and was not altered by elimination of the aldosteroneinduced increase in voltage and short-circuit current with 10 gM amiloride. Studies with inhibitors and ion substitution revealed that mucosal acidification resulted from both Na-dependent and Na-independent mechanisms. Na-dependent acidification was inhibited by ATPase inhibitors and was mediated in part by a luminal Na +/H + exchanger in the presence of secondary hyperaldosteronism. Na-independent acidification was mediated by a pathway dependent on luminal K + that was inhibited by vanadate and mucosal ouabain, consistent with the operation of an H +/K + exchange pump.