Atrioventricular (AV) nodal conduction time is known to be modulated by the autonomic nervous system. The presence of numerous parasympathetic and sympathetic nerve fibres in association with conduction tissue in the heart is well authenticated. In this study, confocal microscopy was used to image the distribution of antibodies directed against the general neuronal marker PGP 9.5, tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and β1 and β2-adrenoreceptors. Serial 12 μm sections of fresh frozen tissue taken from the frontal plane of the rat atrioventricular node, His bundle and bundle branches were processed for histology, acetylcholinesterase (AChE) activity and immunohistochemistry. It was found that the AV and ventricular conduction systems were more densely innervated than the atrial and ventricular myocardium as revealed by PGP 9.5 immunoreactivity. Furthermore, the transitional cell region was more densely innervated than the midnodal cell region, while spatial distribution of total innervation was uniform throughout all AV nodal regions. AChE-reactive nerve processes were found throughout the AV and ventricular conduction systems, the spatial distribution of which was nonuniform exhibiting a paucity of AChE-reactive nerve processes in the central midnodal cell region and a preponderance in the circumferential transitional cell region. TH-immunoreactivity was uniformly distributed throughout the AV and ventricular conduction systems including the central midnodal and circumferential transitional cell regions. β1-adrenoreceptors were found throughout the AV and ventricular conduction systems with a preponderance in the circumferential transitional cell region. β2-adrenoreceptors were localised predominantly in AV and ventricular conduction systems with a paucity of expression in the circumferential transitional cell region. These results demonstrate that the overall uniform distribution of total nerve processes is comprised of nonuniformly distributed subpopulations of parasympathetic and sympathetic nerve processes. The observation that the midnodal cell region exhibits a differential spatial pattern of parasympathetic and sympathetic innervation suggests multiple sites for modulation of impulse conduction within this region. Moreover, the localisation of β2-ARs in the AV conduction system, with an absence of expression in the circumferential transitional cell layer, suggests that subtype-specific pharmacological agents may have distinct effects upon AV nodal conduction.