The diuretic drug amiloride and its 5-amino substitute N5-methyl-N5-propylamiloride (MPA) are potent inhibitors of the growth factor-activatable Na+/H+ exchanger isoform 1 (NHE1). This inhibitor competes with Na+, presumably by interacting with the ion-transport site of the NHE molecule. As an approach to identify this site, we previously reported the use of a specific H(+)-killing selection technique for isolating amiloride-resistant variants of Chinese hamster lung fibroblasts. After long-term selection, two variants, AR40 and AR300, 100- and 1000-fold, respectively, resistant to MPA, were isolated. By comparing NHE1 cDNA sequences of parental and two variant cell lines, we show that the 1000-fold resistance to MPA results from two sequential genetic events. (i) In one AR40 allele a point mutation, Phe-167--> Leu, occurs in the middle of the fourth putative transmembrane segment of NHE1. Producing this mutant protein from human NHE1 cDNA by site-directed mutagenesis increased the Ki for MPA by 30-fold, as seen in AR300 cells. (ii) An approximately 10-fold amplification of the mutated allele, which contributes to the acquired MPA resistance, accounts for the Vmax increase. Mutating a close residue, Phe-165--> Tyr, increased by 40-fold the Ki for amiloride and reduced Na+ transport rate 3- to 4-fold, indicating that we have identified a critical domain of the NHE molecule that controls amiloride binding and Na+ transport. Interestingly, the epithelial amiloride-resistant NHE isoforms that occurred naturally possess some of the amino acid substitutions described here.