This study examines the hypothesis that neuronal infectivity and the spreading of the pseudorabies virus (PRV) through the synapses in the central nervous system (CNS) are influenced by the oestrogen levels. The arcuate nucleus (ARC) and the subfornical organ (SFO) were chosen as models for analysis; the neurons in both structures possess oestrogen receptors and are mutually connected. A genetically engineered pseudorabies virus (Ba-DupLac) was used as a transneuronal tract tracer. This virus is taken up preferably by axon terminals, and transported very specifically through the synapses in a retrograde manner. Ba-DupLac was injected into the ARC of rats, followed by monitoring of the PRV-immunoreactivity (PRV-IR) in the SFO 72 h following inoculation. We found no PRV immunolabelling in the SFO of ovariectomized (OVX) rats, or in those OVX animals that received oestrogen shortly (4 h) before PRV infection (OVX + E 4 h). In contrast, in those OVX animals that received oestrogen 12 h before PRV infection (OVX + E 12 h), and also in intact control animals, PRV-IR was demonstrated in the SFO in all cases. Surprisingly, a reverse labelling was observed in the OVX rats; PRV-IR appeared in the pyriform cortex, whereas PRV-IR could not be detected in the control and OVX + E 12 h animals. As far as we are aware, this is the first study to demonstrate that transneuronal PRV labelling depends on the effects of oestrogen on certain CNS structures and connections.