It is well known that stress inhibits normal reproductive function, including gonadotrophin secretion; however, the mechanisms and mediators involved are largely unknown. Stress impairs the secretion of luteinising hormone (LH), and it has been suggested that the RF-amide gonadotrophin-inhibitory hormone (GnIH), known as RF-amide related peptide-3 (RFRP-3) in mammalian species, may mediate this inhibitory effect of stress. If this is the case, the GnIH/RFRP system would likely be up-regulated during stress. We tested this hypothesis in ovariectomised ewes using a psychosocial stressor: isolation/restraint. Ewes were randomly allocated to control or stress (n = 5 per group). Isolation/restraint stress was imposed for 90 min after control sampling for 4 h, whereas control ewes were sampled continuously for 5.5 h. All ewes were then euthanased and brains were collected. As expected, plasma concentrations of cortisol were increased significantly (P <0.05) by stress and plasma concentrations of LH were significantly (P <0.05) reduced. Immunohistochemistry and in situ hybridisation were conducted for RFRP-3 peptide and RFRP mRNA expression, respectively, in the paraventricular nucleus/dorsal medial hypothalamus region of the hypothalamus. There was no significant effect of stress on RFRP-3 peptide or mRNA levels, with no differences between control or stress ewes. Furthermore, there was no difference in the number of RFRP-3 cells double-labelled for Fos between control and stress ewes and there was no difference in the cellular expression of RFRP mRNA between groups. These results indicate that the GnIH/RFRP system is not activated by psychosocial stress in ewes, suggesting that it is an unlikely mediator of the effects of stress on LH secretion.