The Na⁺ -Ca²⁺ exchanger is the major Ca²⁺ extrusion mechanism in cardiac myocytes. The activity of the cardiac Na⁺-Ca²⁺ exchanger is dynamically regulated by intracellular Ca²⁺. Previous studies indicate that Ca²⁺ binding to a high-affinity Ca²⁺-binding domain (CBD1) in the large intracellular loop is involved in regulation. We generated transgenic zebrafish with cardiac-specific expression of CBD1 linked to YFP and CFP. Ca²⁺-binding to CBD1 induces conformational changes as detected by FRET. With this transgenic fish model, we were able to monitor conformational changes of the Ca²⁺ regulatory domain of NCX in intact hearts. Treatment with the positive inotropic agents ouabain and isoproterenol increased both Ca²⁺ transients and Ca²⁺-induced changes in FRET. The results indicate that Ca²⁺ regulation of the Na⁺-Ca²⁺ exchanger domain CBD1 changes with inotropic state. The transgenic fish models will be useful to further characterize the regulatory properties of the Na⁺-Ca²⁺ exchanger in vivo.