Gonadotropin-releasing hormone (GnRH) secretion is controlled by various factors including the excitatory neurotransmitter glutamate. Estrogen (E) regulates GnRH secretion via E responsive cells in the brain that relay the feedback effects to the preoptic area (POA). We used an antibody to vesicular glutamate transporter 2 (VGluT2) to label glutamatergic neurons in the areas of the ewe brain that control GnRH secretion. VGluT2-immunoreactive cells were observed in the arcuate nucleus (ARC)/ventromedial hypothalamic nucleus (VMH) complex, POA, bed nucleus of stria terminalis (BnST) and A1 and A2 cell groups in the brainstem. In 3 ewes, E receptor-' was detected in 52-61% of glutamatergic neurons in ARC/VMH, 37-52% of neurons in the POA and 37-58% of neurons in the BnST. E injection (im or iv) increased the percentage of glutamatergic cells that expressed Fos protein in the ARC (P<0.01 and P<0.001, respectively). In 6 ewes, injection of the retrograde tracer FluoroGold into the POA labeled cells in the ARC and 6-29% of these were also VGluT2-immunoreactive. Double-labelling of varicosities in the POA showed co-localisation of VGluT2 in 12.5 ' 3 % of dopamine ÃŸ hydroxylase immunoreactive terminals, indicating that a subset of glutamatergic inputs could arise from brain stem noradrenergic neurons cells. In the POA, 60% of GnRH neurons had close appositions that were VGluT2-immunoreactive. We conclude that E responsive glutamatergic neurons arising from the brain stem, the BnST and ARC/VMH provide input to the POA and E may involve these, either directly or indirectly, in the regulation of GnRH secretion.