The interaction of the ubiquitous Na+/H+ exchanger, NHE1, with its commonly used inhibitors, amiloride- and benzoyl-guanidine (HOE)-type compounds, is incompletely understood. We previously cloned NHE1 from Amphiuma tridactylum (atNHE1) and Pleuronectes americanus (paNHE1). While highly homologous to the amiloride- and HOE-sensitive human NHE1 (hNHE1), atNHE1 is insensitive to HOE-type-, and paNHE1 to both amiloride- and HOE–type compounds. Here, we generated chimeras to “knock in” amiloride- and HOE-sensitivity to paNHE1, and thereby identified several NHE1 regions involved in inhibitor interaction. The markedly different inhibitor sensitivities of hNHE1, atNHE1, paNHE1 could not be accounted for by differences in TM9. Replacing TM10 through the C-terminal tail of paNHE1 with the corresponding region of atNHE1 partially restored sensitivity to amiloride and the related compound EIPA, yet not to HOE694. This effect was not due to the tail region, but was dependent on TM10-11, since replacing only this region with that of atNHE1 also partially restored amiloride- and EIPA-, yet not HOE-sensitivity. The converse mutant (TM10-11 of atNHE1 replaced with those of paNHE1) exhibited even higher amiloride- and EIPA-sensitivity, and was also HOE-sensitive. Replacing a LFFFY motif in transmembrane region (TM)4 of paNHE1 with the corresponding residues of hNHE1 (VFFLF) or atNHE1 (TFFLF) greatly increased sensitivity to both amiloride- and HOE-type compounds, although atNHE1 is HOE694-insensitive. Gain of amiloride sensitivity appeared to correlate with increased Na+/H+ exchange rates. It is concluded that regions within TM4 and TM10-11 contribute to amiloride- and HOE-sensitivity, both regions imparting partial inhibitor sensitivity to NHE1.