SLC26A3, a Cl^-/HCO₃^- exchanger exchanger, is highly expressed in intestinal epithelial cells, and its mutations cause congenital chloride diarrhea. This suggests that SLC26A3 plays a key role in NaCl absorption in the intestine. Electroneutral NaCl absorption in the intestine is mediated by functional coupling of the Na⁺/H⁺ exchanger and Cl^-/HCO₃^- exchanger exchanger. It is proposed that the coupling of these exchangers may occur as a result of indirect linkage by changes of intracellular pH (pH_i). We therefore investigated whether SLC26A3 is regulated by pH_i. We generated a hemagglutinin epitope-tagged human SLC26A3 construct and expressed it in CHO cells. Transport activities were measured with a fluorescent chloride-sensitive dye, diH-MEQ. pH_i was clamped at a range of values from 6.0 to 7.4. We monitored the transport activity of SLC26A3 by reverse mode of Cl^-/HCO₃^- exchanger and Cl^-/NO₃^- exchange. None of these exchange modes induced membrane potential changes. At constant external pH 7.4, Cl^-/HCO₃^- exchange was steeply inhibited with pH_i decrease between 7.3 and 6.8 as opposed to thermodynamic prediction. In contrast, however, Cl^-/NO₃^- exchange was essentially insensitive to pH_i within physiological ranges. We also characterized the pH_i dependency of C-terminal truncation mutants. Removal of the entire C-terminal resulted in decrease of the transport activity, but did not noticeably affect pH_i sensitivity. These results suggest that Cl^-/HCO₃^- exchange mode of SLC26A3 is controlled by a pH-sensitive intracellular modifier site, which is likely in the transmembrane domain. These observations raise the possibility that human SLC26A3 activity may be regulated via NHE3 through the alteration of pH_i under physiological conditions.