Eukaryotic Na(+)/H(+) exchangers are transmembrane proteins that are vital for cellular homeostasis and play key roles in pathological conditions such as cancer and heart diseases. Using the crystal structure of the Na(+)/H(+) antiporter from Escherichia coli (EcNhaA) as a template, we predicted the three-dimensional structure of human Na(+)/H(+) exchanger 1 (NHE1). Modeling was particularly challenging because of the extremely low sequence identity between these proteins, but the model structure is supported by evolutionary conservation analysis and empirical data. It also revealed the location of the binding site of NHE inhibitors; which we validated by conducting mutagenesis studies with EcNhaA and its specific inhibitor 2-aminoperimidine. The model structure features a cluster of titratable residues that are evolutionarily conserved and are located in a conserved region in the center of the membrane; we suggest that they are involved in the cation binding and translocation. We also suggest a hypothetical alternating-access mechanism that involves conformational changes.