The Na⁺/Ca²⁺ exchanger family is a member of low-affinity, high-capacity Ca²⁺ transporters, which are driven by membrane potential as well as Na⁺ and Ca²⁺ concentration gradients. The Na⁺/Ca²⁺ exchanger family forms multigene family comprising three groups: plasma membrane Na⁺/Ca²⁺ exchangers (NCX), plasma membrane Na⁺/Ca²⁺/K⁺exchangers (NCKX), and mitochondrial inner membrane Na⁺/Ca²⁺ exchangers (NCLX). Recent structural analyses revealed that the Na⁺/Ca²⁺ exchanger family consists of ten membrane-spanning segments, containing two highly conserved α-repeat regions (α-1, α-2), known to be important for ion binding and translocation. In this study, taking advantage of structural similarity in the Na⁺/Ca²⁺ exchanger family, we conducted chimeric analysis between NCKX2 and NCLX to study the structural domains responsible for the difference in their localization (plasma membrane and mitochondrial inner membrane). Our results using CHO cells expressing several NCKX2-NCLX chimeras suggest that NCKX2 and NCLX may have specific regions that determine its proper placement on plasma membrane and mitochondrial inner membrane, respectively.