Vagal nerve stimulation has been reported to enhance memory in both rats and humans, and to be an effective treatment for epilepsy in some patients, but the underlying neuroanatomical substrate(s) responsible for these effects remains unknown. Since there is no direct anatomical projection from the nucleus tractus solitarius, the main vagal relay site of the brain, to the hippocampus, we tested whether a multisynaptic pathway exists. Pseudorabies virus, a pig herpesvirus that can be used as a retrograde transneuronal tracer, was injected into the ventral CA1 hippocampus of rats, and after 4 days, pseudorabies virus infected neurons were identified in the general visceral portion of the nucleus tractus solitarius, with the majority being localized in the A2 noradrenergic cell group. Other autonomic brainstem nuclei, including the parabrachial nucleus, locus coeruleus, A1 and A5 noradrenergic cell groups, and C1 adrenergic cell group, were labeled. In order to identify some of the potential relay sites of the nucleus tractus solitarius-->hippocampal pathway, immunotoxin lesions of the ventral CA1 region were made that selectively destroyed either the noradrenergic or cholinergic fibers. After 2 weeks' recovery, pseudorabies virus was injected in this same CA1 area, and 4 days later, the transneuronal labeling in the nucleus tractus solitarius was reduced by approximately 65%. These findings suggest that the noradrenergic neurons of the locus coeruleus and cholinergic neurons of the medial septum/diagonal band are likely to be relay sites for this pathway. Other potential linkages are discussed. In summary, this is the first anatomical report to show that the general visceral region of nucleus tractus solitarius is linked via multisynaptic relays to the hippocampus.