The effect of β-adrenergic stimulation on cardiac Na⁺/Ca²⁺ exchange has been controversial. To clarify the effect, we measured Na⁺/Ca²⁺ exchange current (I_NCX) in voltage-clamped guinea pig, mouse, and rat ventricular cells. When I_NCX was defined as a 5 mM Ni²⁺-sensitive current in guinea pig ventricular myocytes, 1 μM isoproterenol apparently augmented I_NCX by ∼32%. However, this increase was probably due to contamination of the cAMP-dependent Cl⁻ current (CFTR-Cl⁻ current, I_CFTR-Cl), because Ni²⁺ inhibited the activation of I_CFTR-Cl by 1 μM isoproterenol with a half-maximum concentration of 0.5 mM under conditions where I_NCX was suppressed. Five or ten millimolar Ni²⁺ did not inhibit I_CFTR-Cl activated by 10 μM forskolin, an activator of adenylate cyclase, suggesting that Ni²⁺ acted upstream of adenylate cyclase in the β-adrenergic signaling pathway. Furthermore, in a low-extracellular Cl⁻ bath solution, 1 μM isoproterenol did not significantly alter the amplitude of Ni²⁺-sensitive I_NCX at +50 mV, which is close to the reversal potential of I_CFTR-Cl. No change in I_NCX amplitude was induced by 10 μM forskolin. When I_NCX was activated by extracellular Ca²⁺, it was not significantly affected by 1 μM isoproterenol in guinea pig, mouse, or rat ventricular cells. We concluded that β-adrenergic stimulation does not have significant effects on I_NCX in guinea pig, mouse, or rat ventricular myocytes.