Heart rate is regulated by the autonomic nervous system but little is known about the pattern of innervation of the pacemaker in the sinoatrial node, or the subpopulations of nerves involved. Therefore in this study the pacemaker was located using electrophysiological methods and the pattern of innervation established by cholinesterase staining. In subsequent experiments, subpopulations of sympathetic, sensory and parasympathetic nerves were identified. Sympathetic nerves were labelled by glyoxylic acid-induced catecholamine fluorescence or an antiserum raised against tyrosine hydroxylase (TH). These experiments showed that the entire sinoatrial node was densely innervated by sympathetic axons, the majority of which were immunoreactive for neuropeptide Y (NPY). There were a few axons which were only immunoreactive for TH. Sensory nerves which were immunoreactive for both substance P (SP) and calcitonin gene-related peptide (CGRP) were also found throughout the sinoatrial node. In the absence of a selective marker for parasympathetic neurons, hearts were extrinsically denervated by placing them in organotypic culture to allow degeneration of extrinsic axons. In this way intrinsic parasympathetic neurons could be characterised. These experiments revealed several distinct populations of parasympathetic nerves which innervated only a small, discrete part of the sinoatrial node. These populations were immunoreactive for NPY, somatostatin (SOM) or vasoactive intestinal peptide (VIP) alone, or SOM combined with NPY, SOM with dynorphin B, and SOM with SP. These results highlight a remarkable difference in the pattern of innervation of the sinoatrial node by the sympathetic and parasympathetic nervous systems. Furthermore the presence of several distinct populations of autonomic cardiac neurons indicates a further complexity in neuronal regulation of heart rate.