The Na+/Ca2+ exchanger (NCX) regulates cardiac contractility by adjusting the amount of Ca2+ inside myocytes. NCX accomplishes this by using the electrochemical gradient of Na+: during each cycle three Na+ ions enter the cell and one Ca2+ ion is extruded against its gradient. In addition to being transported, cytoplasmic Na+ and Ca2+ ions also regulate exchanger activity. The physiological relevance and molecular processes underlying ionic regulation remain unclear. Also unresolved are the events that regulate NCX trafficking to the membrane and its oligomeric state. This is essential information to interpret structure–function data. The full-length exchanger was fused to both CFP and YFP, creating active fluorescent exchangers used in FRET experiments to assess both conformational changes associated with ionic regulation and the oligomeric state of NCX. Electrophysiological characterization demonstrates that these constructs behave similarly to the wild-type (WT) exchanger. We have been able for the first time to monitor conformational changes of the exchanger Ca2+-binding site in vivo. These studies provide a better understanding of the molecular properties of the NCX.