The widely expressed anion exchanger polypeptide AE2/SLC4A2 is acutely inhibited by acidic intracellular (pHi), by acidic extracellular pH (pHo), and by the calmodulin inhibitor, calmidazolium, whereas it is acutely activated by NH4+. The homologous erythroid/kidney AE1/SLC4A1 polypeptide is insensitive to these regulators. Each of these AE2 regulatory responses requires the presence of AE2's C-terminal transmembrane domain (TMD). We have now measured 36Cl− efflux from Xenopus oocytes expressing bi- or tripartite AE2–AE1 chimeras to define TMD subregions in which AE2-specific sequences contribute to acute regulation. The chimeric AE polypeptides were all functional at pHo 7.4, with the sole exception of AE2(1-920)/AE1(613-811)/AE2(1120-1237). Reciprocal exchanges of the large third extracellular loops were without effect. AE2 regulation by pHi, pHo and NH4+ was retained after substitution of C-terminal AE2 amino acids 1120–1237 (including the putative second re-entrant loop, two TM spans and the cytoplasmic tail) with the corresponding AE1 sequence. In contrast, the presence of this AE2 C-terminal sequence was both necessary and sufficient for inhibition by calmidazolium. All other tested TMD substitutions abolished AE2 pHi sensitivity, abolished or severely attenuated sensitivity to pHo and removed sensitivity to NH4+. Loss of AE2 pHi sensitivity was not rescued by co-expression of a complementary AE2 sequence within separate full-length chimeras or AE2 subdomains. Thus, normal regulation of AE2 by pH and other ligands requires AE2-specific sequence from most regions of the AE2 TMD, with the exceptions of the third extracellular loop and a short C-terminal sequence. We conclude that the individual TMD amino acid residues previously identified as influencing acute regulation of AE2 exert that influence within a regulatory structure requiring essential contributions from multiple regions of the AE2 TMD.