Ion transport performed by the Na(+)/Ca(2+) exchanger (NCX) is regulated via its cytosolic Ca(2+)-binding domains, CBD1 and CBD2, which act as sensors for intracellular Ca(2+). Striking differences in the electrostatic potential of the Ca(2+)-bound and Ca(2+)-free forms turn the CBD1 and CBD2 Ca(2+)-binding sites into electrostatic switches similar to those of C(2) domains. Binding of Ca(2+) with high affinity to CBD1 induces a conformational change that is relayed to the transmembrane domain and thereby initiates Na(+)/Ca(2+) exchange. The Ca(2+) concentration at which this conformational change occurs is determined by the Ca(2+) affinities of the strictly conserved CBD1 Ca(2+)-binding sites that are modulated by an adjacent, variable region of CBD2. In contrast, the Ca(2+)-binding properties of CBD2 depend on the isoform and the type of residues in the Ca(2+)-binding sites, encoded by a mutually exclusive exon. This second electrostatic switch, formed by CBD2, appears to be required for sustained Na(+)/Ca(2+) exchange and may allow tailored, tissue-specific exchange activities.