The Ca²⁺-activated Cl current [I_Cl(Ca)] contributes to the repolarization of the cardiac action potential under physiological conditions. I_Cl(Ca) is known to be primarily activated by Ca²⁺ release from the sarcoplasmic reticulum (SR). L-type Ca²⁺ current [I_Ca(L)] represents the major trigger for Ca²⁺ release in the heart. Recent evidence, however, suggests that Ca²⁺ entry via reverse-mode Na⁺/Ca²⁺ exchange promoted by voltage and/or Na⁺ current (I_Na) may also play a role. The purpose of this study was to test the hypothesis that I_Cl(Ca) can be induced by I_Na in the absence of I_Ca(L). Macroscopic currents and Ca²⁺ transients were measured using the whole cell patch-clamp technique in rabbit ventricular myocytes loaded with Indo-1. Nicardipine (10 µM) abolished I_Ca(L) at a holding potential of 75 mV as tested in Na⁺-free external solution. In the presence of 131 mM external Na⁺ and in the absence of I_Ca(L), a 4-aminopyridine-resistant transient outward current was recorded in 64 of 81 cells accompanying a phasic Ca²⁺ transient. The current reversed at 42.0 ± 1.3 mV (n = 6) and at +0.3 ± 1.4 mV (n = 6) with 21 and 141 mM of internal Cl, respectively, similar to the predicted reversal potential with low intracellular Cl concentration ([Cl]_i) (47.8 mV) and high [Cl]_i (1.2 mV). Niflumic acid (100 µM) inhibited the current without affecting the Ca²⁺ signal (n = 8). Both the current and Ca²⁺ transient were abolished by 10 mM caffeine (n = 6), 10 µM ryanodine (n = 3), 30 µM tetrodotoxin (n = 9), or removal of extracellular Ca²⁺ (n = 6). These properties are consistent with those of I_Cl(Ca) previously described in mammalian cardiac myocytes. We conclude that 1) I_Cl(Ca) can be recorded in the absence of I_Ca(L), and 2) I_Na-induced SR Ca²⁺ release mechanism is also present in the rabbit heart and may play a physiological role in activating the Ca²⁺-sensitive membrane Cl conductance.