This investigation was designed to establish the mechanism of sodium and chloride transport in the rat distal colon by determining ion fluxes across isolated mucosa under voltage clamp conditions. The net rates of sodium and chloride absorption in the distal colon of the rat were approximately equal (5.8 +/- 0.3 and 6.9 +/- 0.5 microEq/h X cm2, respectively) and significantly greater than the short circuit current (0.9 +/- 0.1 microEq/h X cm2). Net sodium absorption and net chloride absorption were markedly reduced by the removal of chloride and sodium, respectively, but were not affected by the absence of potassium from the mucosal bathing solution. Both net sodium absorption and net chloride absorption were also significantly inhibited by 1.0 mM amiloride and by 0.1 mM acetazolamide. In contrast, 0.1 mM amiloride and 1.0 mM furosemide did not inhibit either sodium or chloride absorption. These results confirm that electroneutral sodium chloride absorption is the predominant mechanism of sodium and chloride absorption and suggest that parallel ion (Na-H and Cl-HCO3) exchanges, rather than independent electrogenic sodium and chloride transport, coupled sodium-chloride cotransport, or coupled Na-K-2Cl cotransport, are most likely responsible for sodium chloride absorption in this epithelium.