Sodium-calcium exchanger (NCX) plays crucial role in regulation of cardiac calcium homeostasis and provides a key ionic current (INCX) involved into pacemaker activity of sinoatrial cardiomyocytes. However, modulation of INCX by autonomic neurotransmitters, which contribute to regulation of cardiac performance, still remains questionable. The present study provides the first evidence for direct modulation of INCX via muscarinic receptors. In atrial cardiomyocytes of crucian carp (Carassius carassius L.) and rainbow trout (Oncorhynchus mykiss W.) muscarinic agonist carbacholine (Cch, 10-6-10-4M) produced a strong increase of the outward INCX without any changes in the inward INCX. A selective blocker of INCX, KB-R7943 (10-5 M), abolished the basal INCX (both inward and outward current), which was registered in the absence of Cch, as well as the Cch-induced outward INCX. In ventricular myocytes from both studied species even the high concentration of Cch (10-4 M) was completely ineffective due to the putative absence of muscarinic receptors. On the contrary, in the atrial cells muscarinic antagonist atropine (10-6 M), which blocks M-receptors without any subtype selectivity, completely eliminated the response of outward INCX to Cch, but did not affect the basal INCX. A selective antagonist of M2 cholinoreceptors, AF-DX 116 (2×10-7M) and a M3 antagonist, 4-DAMP (10-9 M), decreased Cch-induced current by 84.4% and 16.6% respectively. Pertussis toxin, which irreversibly inhibits Gi-protein coupled to M2 receptors, reduced the Cch-induced INCX by 95%, when applied into the pipette solution. The present results indicate a vectorial cholinergic stimulation of INCX in fish atrial myocytes, predominantly mediated by M2 cholinoreceptors. We conclude that the selective stimulation of the outward component of INCX by M2 cholinoceptors represents a new molecular target for the cardiac parasympathetic innervations, which is present at least in fish myocardium.