Background-Direct 3D analysis (ie, stereotaxic analysis of 3 planes) has shown that the atrioventricular (AV) node (AVN) is continuous with only specialized myocardium of the proximal AV bundle (PAVB) and distal AV bundle (DAVB) or His bundle. The purpose of the present study was to determine whether the PAVB, AVN, and DAVB possess histological features distinct from each other and from the ordinary myocardium. Methods and Results-A protocol that preserves the cytoplasmic and interstitial integrity of the tissue and permits serial sections of the AV junction region to be made in 3 orthogonal planes showed that the PAVE, AVN, and DAVB are characterized by myocardium aggregated into fascicles containing approximate to 8 myofibers. Myofibers within the fascicles are coiled or spiraled about each other; and spiraling is most compact in the PAVE. Collagen encases individual fascicles and segregates primary fascicles into secondary fascicles. Fascicles, and not myofibers. are in parallel array in the PAVE, interwoven in the AVN, and parallel in the DAVB. Narrow junctions of parallel fascicles separate the AVN from the PAVE and DAVB. Myocytes, which are largest in DAVB, possess clear perinuclear regions; thin finger-like end processes, which are most numerous in the AVN; uniform, delicate cross-striations; and intercalated disks, which are broader in the PAVE and form short stacks in the AVN. Sheaves of nerve terminals, including boutons, are as found in skeletal muscle. Conclusions-The PAVE, AVN, and DAVB have distinct histological features. Collagen septation of primary and secondary fascicles presents natural barriers within the tissues and to surrounding myocardium and structures. These findings confirm that the AV junction region contains a specialized conduction system that is anatomically isolated from ordinary myocardium.