The role of intracellular pH (pH(i)) in regulation of AE2 function in Xenopus oocytes remains unclear. We therefore compared AE2-mediated (36)Cl(-) efflux from Xenopus oocytes during imposed variation of extracellular pH (pH(o)) or variation of pH(i) at constant pH(o). Wild-type AE2-mediated (36)Cl(-) efflux displayed a steep pH(o) vs. activity curve, with pH(o(50)) = 6.91 +/- 0.04. Sequential NH(2)-terminal deletion of amino acid residues in two regions, between amino acids 328 and 347 or between amino acids 391 and 510, shifted pH(o(50)) to more acidic values by nearly 0.6 units. Permeant weak acids were then used to alter oocyte pH(i) at constant pH(o) and were shown to be neither substrates nor inhibitors of AE2-mediated Cl(-) transport. At constant pH(o), AE2 was inhibited by intracellular acidification and activated by intracellular alkalinization. Our data define structure-function relationships within the AE2 NH(2)-terminal cytoplasmic domain, which demonstrates distinct structural requirements for AE2 regulation by intracellular and extracellular protons.