Chapter 1 of this thesis introduces Ca2+ signaling and the role of the Na+/Ca2+-exchanger (NCX) in Ca2+ homeostasis. It describes the general features of NCX function and regulation and indicates the importance of the Ca2+-binding domains (CBD1 and CBD2). The subsequent Chapters of this thesis present a detailed analysis of the CBDs using Nuclear Magnetic Resonance (NMR) and other biophysical techniques. Chapter 2 gives a general introduction of NMR as a tool to study proteins. Chapter 3 describes the structural and dynamical effects of Ca2+ binding to CBD2 and the results are compared between splice variants and other Ca2+-binding proteins. NCX1-CBD2-AD has two Ca2+ binding sites and the data show highly similar Ca2+ affinities for both sites, even though the primary site has six coordinating carboxylates and the secondary site only two, indicating positive cooperative binding. The 15N-relaxation data show that Ca2+ binding to CBD2 rigidifies not only loops near the Ca2+ binding sites but also in the FG-loop at the opposite site of the domain. This suggests that the Ca2+-binding event is felt throughout the molecule and implicates an import role for the FG- loop in connecting the Ca2+ binding event of CBD2 with other cytosolic domains of the NCX. Divalent cations, such as Mg2+, compete with Ca2+ for the Ca2+ binding sites. The Mg2+ concentration inside the cell is approximately a thousand times higher than the intracellular Ca 2+ concentration. Therefore, functional Ca2+ sensors must have binding sites highly specific for Ca2+. Chapter 4 shows that only one of the two binding sites of NCX1-CBD2-AD is specific for Ca2+. Mg2+ always occupies the other binding site and the Mg2+-bound NCX1-CBD2-AD is structurally more stable and has an altered Ca2+ affinity for the remaining binding site than apo NCX1-CBD2-AD. A solution structure of NCX3-CBD2-B in the Ca2+ bound form is presented in Chapter 5. This is the first structure of a domain of NCX3. The structure is highly homologous to NCX1-CBD2-AD. The new structure shows that the overall fold of CBD2, including the b-bulges and the a-helix, are conserved between the different NCX isoforms. The orientation of the a-helix with respect to the b-sandwich is determined for the first time using residual dipolar coupling measurements. The relevance of this orientation in the full-length exchanger is discussed. The Calx-b motif is compared to other motifs of the immunoglobulin fold in Chapter 6. A structural explanation is given for the distinct Ca2+ binding properties of the different CBD variants and a gives a short outlook on NCX research. Finally, Chapter 7 presents an overall summary of this thesis.