Absorption of HCO^-₃ in the MTAL is mediated by apical membrane Na⁺/H⁺ exchange (NHE3). The identity and function of other apical acid-base transporters in this segment have not been defined. The present study was designed to examine apical membrane HCO^-₃/OH^-/H⁺ transport pathways in the rat MTAL and determine their role in transepithelial HCO^- ₃ absorption. MTAL were perfused in vitro in Na⁺- and Cl^-- free solutions containing 25 mM HCO^-₃ , 5% CO₂. Lumen addition of either 120 mM Cl^- or 50 mM Na⁺ (50 µM EIPA present) had no effect on intracellular pH (pH_i). Lumen Cl^- addition also had no effect on pH_i in the presence of 145 mM Na⁺ or in the nominal absence of HCO^-₃/CO₂. Thus, there was no evidence for apical Cl^-/HCO^-₃(OH^-) exchange, Na⁺-dependent Cl^-/HCO^-₃ exchange, or Na⁺-HCO^-₃ cotransport. In contrast, in tubules studied in Na⁺- and Cl^- - free solutions containing 25 mM HCO^-₃, 5% CO₂ and 120 mM K⁺, removal of luminal K⁺ induced a rapid and pronounced decrease in pH_i (pH_i = 0.56 ± 0.06 pH units). pH_i recovered following lumen K⁺ readdition. The initial rate of net base efflux induced by lumen K⁺ removal was decreased 85% at the same pH_i in the nominal absence of HCO^-₃/CO₂, indicating a dependence on HCO^-₃/CO₂ and arguing against apical K⁺/H⁺ exchange. A combination of the apical K⁺ channel blockers quinidine (0.1 mM) and glybenclamide (0.25 mM) had no effect on the lumen K⁺- induced pH_i changes, arguing against electrically coupled K⁺ and HCO^-₃ conductances. The effect of lumen K⁺ on pH_i was inhibited by 1 mM H₂DIDS. In addition, lumen addition of DIDS increased transepithelial HCO^-₃ absorption from 10.7± 0.7 to 14.9±0.7 pmol/min/mm (P<0.001) and increased pH_i slightly in MTAL studied in physiological solutions (25 mM HCO^-₃ and 4 mM K⁺). Lumen DIDS stimulated HCO^-₃ absorption in the absence and presence of furosemide. These results are consistent with an apical membrane K⁺-dependent HCO^-₃ transport pathway that mediates coupled transfer of K⁺ and HCO^-₃ from cell to lumen in the MTAL. This mechanism, possibly an apical K⁺- HCO^-₃ cotransporter, functions in parallel with apical Na⁺/H⁺ exchange and opposes transepithelial HCO^-₃ absorption.