Abstract The use of Herpes simplex virus (HSV) as a retrograde transneuronal tracer would have the unique advantage that the virus would be replicated in the second order neurones, resulting in strong labelling. HSV was injected in the XII nerve (mice). The virus was detected immunohistochemically. Four stages in the brainstem distribution of HSV-positive neurones were distinguished. These stages were correlated with injected amounts/survival time. In stage 1, positive neurones were restricted to the XII nucleus; glial cells were present around the intramedullary XII rootlets. In stages 2–4, positive neurones and glial cells were also present outside the XII nucleus: (a) in the lateral reticular formation, Kölliker-Fuse nucleus, raphe and nucleus coeruleus; and (b) in the area around the XII rootlets, including parts of the inferior olive. In view of their distribution, many of the neurones in (a) must have received the virus by retrograde transneuronal transfer from XII motoneurones. The neurones in (b) were probably infected through a different route, i.e. local transfer of virus from XII axons via glial cells. This local transfer does not lead to extensive spread of the infection, yet, when using HSV for retrograde transneuronal tracing it may represent a source of error.