The crystal structures of the CBD1 and CBD2 domains of the Na+/Ca2+ exchanger protein (NCX1) provided a major breakthrough in Ca2+-dependent regulation of NCX1, although the dynamic aspects of the underlying molecular mechanisms are still not clear. Here we provide new experimental approaches for evaluating the kinetic and equilibrium properties of Ca2+ interaction with regulatory sites by using purified preparations of CBD1, CBD2, and CBD12 proteins. CBD12 binds approximately 6 Ca2+ ions (mol/mol), whereas the binding of only approximately 2 Ca2+ ions is observed (with a Hill coefficient of nH=approximately 2) either for CBD1 or CBD2. In the absence of Mg2+, CBD1 has a much higher affinity for Ca2+ (Kd=0.3+/-1.2 microm) than CBD2 (Kd=5.0+/-1.2 microm). The Ca2+ dissociation from CBD2 (koff=230+/-70 s(-1)) is at least 25 times faster than from CBD1 (koff=10+/-3 s(-1)), whereas the kon values indicate fast kinetics for Ca2+ binding (kon=koff/Kd=10(7)-10(8) m(-1) s(-1)) for both CBDs. At 2-5 mm Mg2+, both CBDs bind Ca2+, with a Kd of 1-2 microm (Mg2+ has very little effect on Ca2+ off rates). Mg2+ cannot occupy the primary site of CBD2, whereas the other Ca2+ sites of CBDs interact with Mg2+ as well. There is no competition between Na+ and Ca2+ for any CBD site. The kinetically diverse Ca2+ sensors may sense differentially the dynamic swings in [Ca2+] within specific subcellular compartments (dyadic cleft, submembrane space, bulk cytosol, etc.).