The colon is believed to absorb NaCl via the coupled operation of apical Na/H exchanger-3 (NHE3) and Cl/HCO(3) exchanger SLC26A3 (DRA). Efficient coupling requires that NHE3 and DRA operate in close proximity within common luminal and cytosolic microenvironments. Thus we examined whether these proteins coexist along the apical margin of surface enterocytes by quantitative immunofluorescence microscopy in consecutive colon segments from nonfasted mice and rats. The cecocolonic profiles of NHE3 and DRA expression were roughly inverse; NHE3 was highest in proximal colon (PC) and negligible in distal colon, whereas DRA was absent in early PC and highest in the late midcolon, and DRA was prominent in the cecum whereas NHE3 was not. NHE3 and DRA coexisted only in the middle third of the colon. The consequences of unpaired NHE3/DRA expression on mucosal surface (subscript MS) pH and Na(+) concentration ([Na(+)]) were assessed in nonfasted rats in situ using miniature electrodes. In the cecum, where only DRA is expressed, pH(MS) was approximately 7.5, markedly higher than underlaying stool (6.3), consistent with net HCO(3)(-) secretion. In the early PC, where NHE3 is not expressed with DRA, pH(MS) was acidic (6.2), consistent with unopposed H(+) secretion. [Na(+)](MS) was approximately 60 mM in the cecum, decreased along the PC to approximately 20 mM, and declined further to approximately 10 mM distally. Cl(-) was secreted into the PC, then reabsorbed distally. Our results suggest a model in which 1) unpaired DRA activity in the cecum maintains an alkaline mucosal surface that could neutralize fermentative H(+); 2) unpaired NHE3 activity in the early PC preserves an acidic mucosal surface that could energize short-chain fatty acid absorption; and 3) coupled NHE3/DRA activities in the midcolon allow for vigorous NaCl absorption at a neutral pH(MS).