Background: The sinoatrial node (SAN) is the primary pacemaker of the heart. It is a heterogeneous structure – for example, the centre of the SAN is the leading pacemaker site, whereas the periphery of the SAN is specialised to drive the surrounding atrial muscle. Current knowledge of SAN morphology is limited. Here for the first time we apply 3-D imaging techniques, serial block face scanning electron microscopy (SBF-SEM), to examine the ultrastructure of the SAN cells to investigate (i) the nano-architecture and (ii) the mode of interaction and communication with adjacent atrial cells. Methods: Hearts were taken immediately after rabbits were killed with an overdose of pentobarbitone (100 mg/kg iv). The SAN region was dissected, and 1 x 1 mm tissue pieces were obtained from the centre and the periphery. The samples were then processed for SBF-SEM as described previously (1). SBF-SEM was carried out on the FEI Quanta 250 FEG SEM equipped with a Gatan 3View system. Images were scanned at 4096×4096 pixels leading to a final resolution of 20 nm/pixel. The cut thickness was set to 50 nm so the voxel size was 20; 20; 50 nm in the X; Y; Z directions respectively. The images were segmented using IMOD (2). Results: At the leading pacemaker site, we found that the SAN contained isolated and clustered SAN cells, fibroblasts, abundant wavy collagen fibres, sparsely distributed elastin fibres, nerve fibres running alongside clusters of SAN cells and a few ganglion cells. The SAN cells contained many glycogen granules. The cells were found to be mono- and bi-nucleated. Our new 3-D structures have revealed that they exhibit spindle or spider-like shapes with projections of various lengths and widths. Due to the lack of regular sarcomeric structures and disorganised mitochondria the cells appeared to have regions that are ‘empty’. SAN cells widely contacted each other via invaginated plasma membrane. Neighbouring cells were also connected via gap junctions. In the peripheral region, SAN cells abutted atrial myocytes. Segmentation of a nodal cell and the neighbouring atrial cell revealed a novel organisation with a protuberance from the SAN cell wrapping around an atrial myocyte with an ultrastructure typical of a working myocyte. Conclusion: Here we show for the first time the nano-architecture of SAN cells, revealing that they form a meshwork with various other cell types surrounded by dense fibrous tissue. Applying the SBF-SEM technique has provided new insights into how SAN cells communicate with each other and with atrial myocytes.