The recent introduction of live viruses as intra-axonal tracing agents has raised questions concerning which central neurons are transneuronally labelled after application of the virus to peripheral organs or peripheral nerves. Since the central connections of the vagus nerve have been well described using conventional neuronal tracing agents, we chose to inject Herpes Simplex Virus Type 1 into the cervical vagus of the rat. After survival times of up to 3 days the rat brains were processed immunohistochemically using a polyclonal antiserum against herpes simplex virus. Two days after injection of the virus we observed viral antigen in the area postrema and in the nucleus tractus solitarius and the dorsal motor nucleus of the vagus (dorsal vagal complex), principally ipsilaterally. At this survival time the viral antigen in the dorsal vagal complex was largely confined to glial cells. After 3 days the viral antigen was localized both in glia and in nerve cells within the dorsal vagal complex and in brain regions previously demonstrated, using conventional tracing procedures, to contain neurons with axonal projections to the dorsal vagal complex. This was true for medullary, pontine, midbrain and hypothalamic regions and for telencephalic regions including the amygdala, the bed nucleus of the stria terminalis, and the insular and medial frontal cortices. Many of the nerve cells containing viral antigen were displayed in a Golgi-like manner, with excellent visualization of the dendritic tree. Axonal processes, in contrast, were not visualized. We used co-localization studies to confirm previous findings concerning monoamine neurotransmitter-related antigens present in medullary and pontine neurons projecting to the dorsal vagal complex. After 3 days there were many Herpes Simplex Virus Type 1-containing glial cells along the intramedullary course of the vagal rootlets. However, no viral antigen was found in brain regions containing neurons whose axons pass through the region of glial cell-labelled rootlets. Glial cells containing viral antigen were particularly numerous in brain regions known to receive an input from neurons in the area postrema and the dorsal vagal complex. Taken together with our observation concerning the early appearance of viral antigen within glial cells in the dorsal vagal complex, this suggests that when the virus reaches the axon terminal portion it is transferred to nearby glial cells and possibly enters central neurons by way of these structures.