Mammalian Na/H exchanger type I isoform (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH (pHi) by removing one intracellular proton in exchange for one extracellular sodium ion. Abnormal activity of the protein occurs in cardiovascular disease and breast cancer. The purpose of this study is to examine the role of negatively charged amino acids of extracellular loop 3 (EL3) in the activity of the NHE protein. We mutated glutamic acid 217 and aspartic acid 226 to alanine, and to glutamine and asparagine, respectively. We examined effects on expression levels, cell surface targeting and activity of NHE1, and also characterized affinity for extracellular sodium and lithium ions. Individual mutation of these amino acids had little effect on protein function. However, mutation of both these amino acids together impaired transport, decreasing the Vmax for both Na and Li ions. We suggested that amino acids E217 and D226 form part of a negatively charged coordination sphere, which facilitates cation transport in the NHE1 protein.