Activity of the AE2/SLC4A2 anion exchanger is modulated acutely by pH, influencing the transporter's role in regulation of intracellular pH and epithelial solute transport. In Xenopus oocytes, heterologous AE2-mediated Cl^-/Cl^- and Cl^-/HCO₃^- exchange is inhibited by acid pH_i or pH_o. We have investigated the importance to pH-sensitivity of the eight histidine (His) residues within the AE2 C-terminal transmembrane domain (TMD). Wildtype (wt) mouse AE2 (mAE2)-mediated Cl^-/Cl^- exchange, measured as DIDS-sensitive ³⁶Cl^- efflux from Xenopus oocytes, was experimentally modulated either by varying pH_i at constant pH_o, or by varying pH_o. Pretreatment of oocytes with the His modifier diethylpyrocarbonate (DEPC) reduced basal ³⁶Cl^- efflux at pH_o 7.4 and acid-shifted the pH_o vs. activity profile of wt-AE2, suggesting the possible involvement of His residues in pH-sensing. Single His mutants of AE2 were generated and expressed in oocytes. Although mutation of H1029 to alanine severely reduced both transport and surface expression, other individual His mutants exhibited wt or near-wt levels of Cl^- transport activity with retention of pH_o sensitivity. But, in contrast to the effects of DEPC on wt-AE2, pH_o-sensitivity was significantly alkaline-shifted for mutants H1144Y, H1145A, and the triple mutants H846/H849/H1145A -and H846/H849/H1160A. While all functional mutants retained sensitivity to intracellular pH, pH_i sensitivity was enhanced for AE2 H1145A. The simultaneous mutation of five or more His residues, however, greatly decreased basal AE2 activity, consistent with the inhibitory effects of DEPC modification. The results show that multiple TMD His residues contribute to basal AE2 activity and its sensitivity to pH_i and pH_o.