Studies were performed to determine the mechanism by which short-chain fatty acids increase colonic Na and Cl absorption by determining unidirectional 22Na and 36Cl fluxes across isolated stripped mucosa from the rat distal colon under voltage clamp conditions. Mucosal butyrate (25 mM, in the absence of bicarbonate) significantly enhanced both net Na and net Cl absorption by 7.0 +/- 1.3 and 6.9 +/- 1.0 microEq/h.cm2, respectively, without increasing the short-circuit current. Net Na and Cl absorption in butyrate-Ringer's solution and HCO3-Ringer's solution were identical. Butyrate stimulation of Na (and Cl) absorption was Cl-dependent and prevented by 1 mM mucosal amiloride, an inhibitor of Na-H exchange, but was HCO3-independent and not inhibited by acetazolamide, a carbonic anhydrase inhibitor. In contrast, bicarbonate-stimulated Na (and Cl) absorption was also Cl-dependent and amiloride-sensitive, but was significantly inhibited by acetazolamide. The effect of mucosal butyrate on net Na and Cl absorption was substantially greater than serosal butyrate, which in the presence of bicarbonate did not alter ion transport. The stimulation of Na and Cl absorption by mucosal butyrate was significantly greater than by propionate and acetate, whereas mucosal formate did not alter Na transport. The results of this study permit the following model: short-chain fatty acid stimulation of active Na and Cl absorption involves uptake of the nonionized form of butyrate and the coupling of Na-H and Cl-butyrate exchanges.