Using immunocytochemical techniques we have examined the distribution of cells containing estrogen receptors (ERs) in the preoptic and anterior hypothalamic regions of short-term (1 week) ovariectomized ewes. Subsequent double-labelling experiments examined the co-localization patterns of ER and luteinizing hormone-releasing hormone (LHRH) or glutamic acid decarboxylase (GAD) immunoreactivities. ER-immunoreactive (-IR) cells were identified throughout the central and medial aspects of the preoptic area in a continuum which begins at the level of the organum vasculosum of the lamina terminalis and terminates in the caudal anterior hypothalamic area. A conspicuous sub-population of densely clustered ER-IR cells was identified within this distribution extending from the central region of the preoptic area into the bed nucleus of the stria terminalis. ER-IR cells were also identified in the ventrolateral septum and supraoptic nuclei. Double-labelling experiments showed that although rostral LHRH neurons were surrounded by ER-IR cells, they did not themselves possess ER immunoreactivity. In marked contrast, we estimate that approximately 40% of GAD-IR cells in the central aspect of the medial preoptic area are immunoreactive for the ER and that these cells account for nearly 30% of all ER-IR cells in this region. These results indicate that, in common with other species, LHRH neurons in the ewe do not possess ERs and suggest therefore, that these neurons are unlikely to be modulated directly by circulating estrogens. However, large numbers of adjacent GABA neurons possess ERs and may comprise a major neuronal population mediating gonadal steroid input to LHRH neurons.