There is conflicting evidence regarding modulation of the Na+-Ca2+ exchanger (NCX) by the β-adrenergic/protein kinase A (PKA) pathway (1). One issue that has been raised is whether or not apparent β-adrenergic stimulation of the NCX in electrophysiological experiments may be confounded by activation of a PKA-dependent Cl- current, carried by cystic fibrosis transmembrane conductance regulator (CFTR) channels (2). The aim of the present study was to establish if activation of the β-adrenergic/PKA pathway stimulates NCX current (INCX) in rabbit ventricular myocytes independent of any Cl- current (ICl,PKA) modulation. We used whole cell patch-clamp experiments at 37° C in ventricular myocytes isolated from rabbit hearts, excised following humane animal-killing in accord with UK Home Office legislation. Recording conditions were used that have been considered specific either for INCX or ICl,PKA. The effects of β-adrenoceptor/PKA activation were studied using 1 μM isoprenaline (ISO), 1 μM noradrenaline (NA) and 10 μM forskolin. INCX was measured as the 10 mM Ni2+- sensitive difference current with other overlapping currents blocked. Under NCX recording conditions, ISO activated a current that was Ni2+-sensitive (n=7). However, the Cl- current activated by each of ISO (n=4) and NA (n=5) was also sensitive to Ni2+, suggesting a contribution of Cl- current to the β-adrenoceptor-activated Ni2+-sensitive current under NCX-recording conditions. On the other hand, the Cl- current activated by forskolin was insensitive to Ni2+ (n=6). Thus, Ni2+ could be used to discriminate between the NCX and Cl- currents activated by forskolin but not by ISO or NA. While the current activated by forskolin in NCX-“selective” conditions was Ni2+ sensitive, there was no significant difference in the current-voltage relations of the Ni2+-sensitive current in control and the Ni2+-sensitive current in forskolin (n=5). These data constitute evidence that in rabbit ventricular myocytes, Ni2+-sensitive current activated by forskolin represents a reliable measure of INCX, without contamination from an overlapping PKA-sensitive Cl- current whereas currents activated by β-adrenoceptor stimulation with ISO/NA in similar conditions are likely a combination of both INCX and CFTR Cl- current both of which are sensitive to Ni2+. Moreover, consistent with a previous independent study (3), the present results indicate that rabbit ventricular INCX is not stimulated by acute β-adrenergic/PKA-activation.