The mechanism by which testicular hormones exert a negative feedback action is unclear, as GnRH neurons do not contain receptors for androgen or oestrogen. The RF-amides, Kisspeptin and RF-amide related peptide-3 (RFRP-3) could be potential neuronal pathways. In ewes, 93% of arcuate kisspeptin cells co-expressed ERα (1), with hypothalamic RFRP-3 cells expressing ERα ranging between 20% in mice (2) and 40% in Syrian hamsters (3). This study aimed to determine if castration influenced the expression of ERα in kisspeptin and RFRP-3 neurons in the ram. Dual label fluorescence immunohistochemistry for the co-expression of the RF-amides with ERα was used to compare the percentage of RF-amide cells containing ERα in the hypothalamus of intact merino rams and long term wethers (n=4)/group, and in rams castrated 4 weeks previously or sham castrated rams, with ewe tissue (luteal phase) included for comparison (n=4/group). Ninety percent of kisspeptin cells expressed ER a in the caudal arcuate nucleus in wethers (long and short term) and ewes. Rams, by comparison, expressed very few kisspeptin cells, and these did not express ERα. Less than 1% of RFRP-3 neurons co-expressed ERα in the merino sheep regardless of group. By contrast, RFRP-3 fibres were in great abundance in intact rams. This suggests that kisspeptin expression and its co-expression with ERα is influenced by testicular hormones. The lack of co-expression of RFRP-3 and ERα in the ram suggests that oestrogen negative feedback in these animals is unlikely to involve RFRP-3 neurons.