Allosteric activation of NCX involves the binding of cytosolic Ca(2+) to regulatory domains CBD1 and CBD2. Previous studies with isolated CBD12 and full-size NCX identified synergistic interactions between the two CBD domains that modify the affinity and kinetic properties of Ca(2+) sensing, although it remains unclear how the Ca(2+)-binding signal is decoded and propagates to transmembrane domains. Biophysical analyses (X-ray, SAXS, and stopped-flow techniques) of isolated preparations of CBD1, CBD2, and CBD12 have shown that Ca(2+) binding to Ca3-Ca4 sites of CBD1 results in interdomain tethering of CBDs through specific amino acids on CBD1 (Asp499 and Asp500) and CBD2 (Arg532 and Asp565). Mutant analyses of isolated CBDs suggest that the two-domain interface governs Ca(2+)-driven conformational alignment of CBDs, resulting in slow dissociation of Ca(2+) from CBD12, and thus, it mediates Ca(2+)-induced conformational transitions associated with allosteric signal transmission. Specifically, occupation of Ca3-Ca4 sites by Ca(2+) induces disorder-to-order transition owing to charge neutralization and coordination, thereby constraining CBD conformational freedom, rigidifying the NCX1 f-loop, and triggering allosteric signal transmission to the membrane domain. The newly found interdomain switch is highly conserved among NCX isoform/splice variants, although some additional structural motifs may shape the regulatory specificity of NCX variants.