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The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitous membrane protein that exchanges one intracellular H+ for an extracellular Na+, thereby regulating cell pH and volume. NHE1 catalytic activity is mediated by a transmembrane (TM) domain with 12 transmembrane segments. We performed cysteine scanning mutagenesis on TMVI (Asn227–Ile249) of NHE1. Each residue of TMVI was mutated into a cysteine in the background of a cysteineless NHE1 protein. MTSET and MTSES are sulfhydryl reactive membrane impermeable compounds able to react with accessible cysteines. Asp238Cys, Pro239Cys, and Glu247Cys expressed inactive NHE1. Asn227Cys, Ile233Cys, and Leu243Cys were strongly inhibited by MTSET, suggesting their pore lining properties. More mutations were introduced to characterize critical residues in TMVI. The Glu248Gln and Leu243Ala mutants were more susceptible to limited proteolytic attack by trypsin suggesting an altered conformation. The results suggest that Glu248 and Leu243 are important in protein structure, stability, and folding.