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Introduction The parasympathetic innervation and cholinergic regulation of the heart is already established before the birth. However, it is unknown how acetylcholine regulates the bioelectrical activity of the superior vena cava’s (SVC) myocardium which demonstrates both pacemaker and working myocardium characteristics during early postnatal period. Materials and Methods Electrically evoked (EAP), spontaneous action potentials (SAP) and resting membrane potential (RPM) were recorded in multicellular SVC preparations of neonatal male Wistar rats using standard microelectrode technique under control conditions and application of acetylcholine (ACh, 1 μM) or a gap junction uncoupling agent octanol (1.2 mM). The data is presented as mean±SD. Results The application of ACh had no effect on RMP of both EAP and SAP, did not alter the slow diastolic depolarization phase of SAP, but induced a significant decrease of the fast sodiumcurrent-dependent upstroke velocity (by 39.8±26.5 V/s, p<0.05) in SAP and prolonged the time delay for EAP excitation (by 0.8±0.5 ms, p<0.05). These ACh effects can be associated with gap junctions uncoupling in SVC. However, the octanol effects were only partially similar with ACh, since octanol induced a decrease of upstroke velocity both in SAP and EAP (by 60.9±30.4 V/s, p<0.05), depolarized the RMP of SAP (by 3±2 ms, p<0.05) and prolonged the EAP activation time delay (by 3.5±2.6 ms, p<0.05). Conclusions The comparison of ACh and octanol effects in neonatal SVC myocardium allow us to suppose that gap junctions and fast sodium current channels are the non-canonical targets for the cholinergic regulation of supraventricular myocardium.