SLC26A3, a Cl(-)/HCO(3)(-) 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(-)/HCO(3)(-) 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 Chinese hamster ovary cells. Transport activities were measured with a fluorescent chloride-sensitive dye dihydro-6-methoxy-N-ethylquinolinium iodide (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(-)/HCO(3)(-) and Cl(-)/NO(3)(-) exchange. None of these exchange modes induced membrane potential changes. At constant external pH 7.4, Cl(-)/HCO(3)(-) exchange was steeply inhibited with pH(i) decrease between 7.3 and 6.8 as opposed to thermodynamic prediction. In contrast, however, Cl(-)/NO(3)(-) exchange was essentially insensitive to pH(i) within physiological ranges. We also characterized the pH(i) dependency of COOH-terminal truncation mutants. Removal of the entire COOH-terminal resulted in decrease of the transport activity but did not noticeably affect pH(i) sensitivity. These results suggest that Cl(-)/HCO(3)(-) exchange mode of human SLC26A3 is controlled by a pH-sensitive intracellular modifier site, which is likely in the transmembrane domain. These observations raise the possibility that SLC26A3 activity may be regulated via Na(+)/H(+) exchanger 3 (NHE3) through the alteration of pH(i) under physiological conditions.