The mechanism for myocardial automaticity may differ among different regions of the heart. In this study, we performed fluorescent ion measurements in cardiomyocytes from the sinoatrial node (SAN), the orthotopic pacemaker, and the pulmonary vein (PV), a potential ectopic pacemaker which may cause atrial fibrillation, focusing on the role of the Na⁺/Ca²⁺ exchanger (NCX). Isolated cardiomyocytes from the guinea pig PV and SAN showing automaticity were loaded with the Ca²⁺ indicators Fluo-4 or Indo-1 for high-speed Ca²⁺ imaging. Inhibition of NCX either by SEA0400 or by low Na⁺ solution decreased the Ca²⁺ transient frequency in PV, but not in SAN. The basal cytoplasmic Ca²⁺ concentration, as well as the number of Ca²⁺ sparks between Ca²⁺ transients, were slightly higher in PV than in SAN. Intracellular Na⁺ concentration, measured by a Na⁺ indicator SBFI, was not different between PV and SAN. The equilibrium potential of NCX (E_NCX) was estimated to be less negative in PV than in SAN. In conclusion, NCX is involved in spontaneous activity in PV, but not in SAN. This is probably because the less negative E_NCX and the more negative voltage range for diastolic depolarization in the PV cause a larger driving force for NCX. In the SAN, whose diastolic depolarization largely overlaps with the E_NCX, the role of NCX in automaticity is limited.