The viral transneuronal tracing method was used to identify the CNS cell groups that regulate the parasympathetic and sympathetic outflow systems of the pancreas. Pseudorabies virus (PRV) was injected into the pancreas of vagotomized rats and after 6 days survival, the pattern of transneuronal labeling in the CNS sympathetic regulatory regions was determined. The converse experiment was performed in order to elucidate the central parasympathetic cell groups that regulate the pancreas. Immunohistochemical methods were used to identify putative neuropeptide- and catecholamine-containing CNS neurons involved in these regulatory circuits. The major finding of this study indicates that five brain regions, viz., paraventricular hypothalamic nucleus, perifornical hypothalamic region, A5 catecholamine cell group, rostral ventrolateral medulla, and lateral paragigantocellular reticular nucleus, contain a considerable amount of overlap in cell body labeling. In addition, the ventrolateral part of the periaqueductal gray matter and gigantocellular reticular nucleus, ventral part also showed a similar overlap, but the numbers of neurons found in these areas were considerably lower than the five major regions. These data suggest that these brain regions may provide parallel and possibly redundant, autonomic pathways affecting glucagon and adrenaline release.