We have previously demonstrated that, in the absence of Na+ in vitro, the rate of colonic K+ absorption is increased by increasing PCO2. Chronic secondary hyperaldosteronism induced by dietary Na-depletion further stimulated K+ absorption under these conditions. Because the observed increments in CO2-dependent K+ absorption were not accompanied by corresponding changes in short-circuit current, macroscopic electroneutrality must have been maintained either by anion absorption or by cation secretion. Colonic Cl- absorption is known to respond to increased PCO2 both in vivo and in vitro, but its response under Na-free conditions and the relationship to K+ absorption have not been examined. To determine the relationship of Cl- absorption to K+, we measured unidirectional fluxes of 36Cl and the response to PCO2 in voltage-clamped segments of rat distal colon. Our findings indicate that the rate of Cl- absorption is increased by increasing CO2, both in the presence and absence of Na+. Under Na-free conditions, Cl- absorption is inhibited by acetazolamide and by the absence of K+;K+ absorption (86Rb or 42K flux) is inhibited in a reciprocal fashion by the absence of Cl-. The rates of K+ and Cl- absorption are similar in controls and after secondary hyperaldosteronism due to a Na-deficient diet. These findings suggest that K- and Cl- absorption are closely coupled under Na-free conditions, most likely due to the operation of parallel, aldosterone-responsive H(+)-K+ and Cl(-)-HCO3- exchange pathways.