The vagal post-ganglionic control of cardiac rate is mediated by two intracardiac ganglia; i.e. the sinoatrial (SA) and posterior atrial (PA) ganglia. Nothing is known about the vagal pre-ganglionic neurons (VPNs) which innervate the PA ganglion, or about the neurochemical anatomy of central afferents which innervate these VPNs. These issues were examined using light microscopic retrograde labeling methods, and dual labeling electron microscopic histochemical and immunocytochemical methods. VPNs projecting to the PA ganglion are found in a narrow column exclusively in the ventrolateral nucleus ambiguus (NA-VL). These neurons are relatively large (37.6 ± 2.7 um by 21.3 ± 3.4 um) with abundant cytoplasm and intracellular organelles, rare somatic and dendritic spines, round uninvaginated nuclei, and myelinated axons. Previous physiological data indicated that microinjections of Neuropeptide Y (NPY) into the NA-VL cause negative chronotropic effects. The present morphological data demonstrate that NPY immunoreactive nerve terminals formed 18 ± 4% of the axo-dendritic or axo-somatic synapses and close appositions upon VPNs projecting to the PA ganglion. Three approximately equal populations of VPNs in the NA-VL were retrogradely labeled from the SA and PA ganglia. One population each projects to the SA ganglion, the PA ganglion, or to both the SA and PA ganglia. Therefore, there are both shared and independent pathways involved in the vagal pre-ganglionic controls of cardiac rate. These data are consistent with the hypothesis that the central and peripheral parasympathetic controls of cardiac rate are coordinated by multiple potentially redundant and/or interacting pathways and mechanisms.