The cardiac Na+/Ca2+ exchanger (NCX1.1) serves as the primary means of Ca2+ extrusion across the plasma membrane of cardiomyocytes after the rise in intracellular Ca2+ during contraction. The exchanger is regulated by binding of Ca2+ to its intracellular domain, which contains two structurally homologous Ca2+ binding domains denoted as CBD1 and CBD2. NMR and x-ray crystallographic studies have provided structures for the isolated CBD1 and CBD2 domains and have shown how Ca2+ binding affects their structures and motional dynamics. However, structural information on the entire Ca2+ binding domain, denoted CBD12, and how binding of Ca2+ alters its structure and dynamics is more limited. Site-directed spin labeling has been employed in this work to address these questions. Electron paramagnetic resonance measurements on singly labeled constructs of CBD12 have identified the regions that undergo changes in dynamics as a result of Ca2+ binding. Double electron-electron resonance (DEER) measurements on doubly labeled constructs of CBD12 have shown that the β-sand-wich regions of the CBD1 and CBD2 domains are largely insensitive to Ca2+ binding and that these two domains are widely separated at their N and C termini. Interdomain distances measured by DEER have been employed to construct structural models for CBD12 in the presence and absence of Ca2+. These models show that there is not a major change in the relative orientation of the two Ca2+ binding domains as a result of Ca2+ binding in the NCX1.1 isoform. Additional measurements have shown that there are significant changes in the dynamics of the F-G loop region of CBD2 that merit further characterization with regard to their possible involvement in regulation of NCX1.1 activity. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.