We have produced transgenic mice which overexpress cardiac Na⁺-Ca²⁺ exchange activity. Overexpression has been assessed by Western blot, Northern blot, and immunofluorescence. Functional overexpression was analyzed using membrane vesicles and isolated ventricular myocytes. In whole cell clamped myocytes dialyzed with 0.1–0.2 mM Fura-2, the magnitude of I_Ca and Ca²⁺_i -transient triggered by I_Ca or caffeine were not significantly different in transgenic vs. control myocytes. In transgenic myocytes, activation of I_Ca, however, was followed by a large slowly inactivating transient inward current representing I_Na-Ca. This current depended on Ca²⁺ release as it was abolished when sarcoplasmic reticulum (SR) Ca²⁺ was depleted using thapsigargin. Ca_i-transients triggered by rapid application of 5 mM caffeine, even though equivalent in control and transgenic myocytes, activated larger I_Na-Ca (∼5 pA/pF at −90 mV) in transgenic vs. control myocytes (1.5 pA/pF). The decay rate of caffeine-induced Ca²⁺_i -transient and I_Na-Ca was 2.5 times faster in transgenic than in control myocytes. 5 mM Ni²⁺ was equally effective in blocking I_Na-Ca in control or transgenic myocytes. In 9 out of 26 transgenic myocytes, but none of the controls, Ca²⁺ influx via the exchanger measured at +80 mV caused a slow rise in [Ca²⁺]_i triggering rapid release of Ca²⁺ from the SR. SR Ca²⁺ release triggered by the exchanger at such potentials was accompanied by activation of transient current in the inward direction. In 2 mM Fura-2–dialyzed transgenic myocytes caffeine-triggered Ca_i-transients failed to activate I_Na-Ca, even though the kinetics of inactivation of I_Ca slowed significantly in caffeine-treated myocytes. In 0.1 mM Fura-2–dialyzed transgenic myocytes 100 μM Cd²⁺ effectively blocked I_Ca and suppressed Ca_i-transients at −10 or +50 mV. Our data suggests that in myocytes overexpressing the exchanger, the content of intracellular Ca²⁺ pools and the signaling of its release by the Ca²⁺ channel vis-à-vis the Na⁺-Ca²⁺ exchanger were not significantly altered despite an up to ninefold increase in the exchanger activity. We conclude that the exchanger remains functionally excluded from the Ca²⁺ microdomains surrounding the DHP/ryanodine receptor complex.