To provide the first description of the exact location of primary pacemaker of the squamate heart, we used sharp microelectrode impalements and optical mapping of isolated sinus venosus preparations from Burmese pythons. We located the dominant pacemaker site at the base of the right leaflet of the sinoatrial valve (SAV), but latent pacemakers were also identified in a circular region around the SAV. Acetylcholine (10−5 mol l−1) or noradrenaline (10−6 mol l−1) induced shifts of the leading pacemaker site to other points near the SAV. The ionic currents of most of the cardiomyocytes isolated enzymatically from the SAV region resembled those of typical working myocytes from the sinus venosus. However, seven cells lacked the background inward rectifier current (IK1) and had a time-dependent hyperpolarization-induced inward current identified as the ‘funny’ pacemaker current (If). Therefore, the region proximal to SAV demonstrates pacemaking activity and contains cells that resemble the electrophysiological properties of mammalian pacemaker myocytes.