Serotonin is involved in female sexual behaviour in which the medial preoptic area (MPA) has a pivotal role. The present study used immunohistochemistry, in situ hybridization and retrograde transport analysis to investigate whether serotonin neurones in the dorsal raphe nucleus (DRN) of females projecting into the MPA contained oestrogen receptor alpha or beta. The projection of serotonin neurones from the DRN to the MPA was confirmed using the microinjection of Fluoro-Gold (FG), a fluorescent retrograde tracer, into the MPA of ovariectomized (OVX-group) and OVX-rats treated with oestradiol benzoate (E2-group). A number of serotonin neurones in the DRN were labelled with FG, indicating that these serotonin neurones in DRN project their terminals into the MPA. FG-labelled serotonin neurones expressed ER beta mRNA in the DRN, and the number of the serotonin neurones containing ER beta mRNA between the OVX-group and the E2-treated group was not significantly different. Serotonin neurones in the DRN did not express ER alpha -immunoreactivity. Since previous findings showed that the density of serotonin-immunoreactive fibres and the concentration of serotonin within the MPA was significantly lower in the E2-group than the OVX-group, our present observations suggested that the regulatory effects of E2 on the serotonergic neurone system in the MPA may be via ER beta within the serotonin-containing cells in the DRN of female rats.