The subcellular mechanism of cell-to-cell communication in the natural pacemaker region of the mammalian heart was studied using electrophysiological and immunofluorescence techniques in isolated pairs of rabbit sinus nodal cells. By measuring whole-cell currents using a double patch-clamp approach, it was demonstrated that communication in the sinus node is mediated through gap junctional channels similar to those in other types of adult cardiac cell pairs. Macroscopic sinus nodal junctional resistance had a mean value of 387.9±97.1 MΩ (mean ±SEM,n=10) and was greatly increased by superfusion with alkanols. Single-channel junctional conductance could be resolved in three cell pairs. Given their high membrane resistance (1.16±0.32 GΩ,n= 12), the electrical coupling provided by as few as three gap junctional channels between nodal cells will allow for pacemaker synchronization. Further evidence for the presence of the channels was obtained from immunofluorescent double-labeling of desmin and the gap junction protein (connexin43) in sinus nodal tissue as well as in cultured sinus nodal cells. Using antisera against residues 243–257 of the connexin43 protein, a specific staining at the site of cell-to-cell apposition was demonstrated. These data provide direct evidence in favor of electrotonic coupling as the means for achieving pacemaker synchronization in the rabbit sinus node.