Previous studies have shown that oestradiol (E₂) decreases the orexigenic effect of melanin-concentrating hormone (MCH). In the present study, we examined whether this action of E₂ is mediated by its ability to decrease the expression of MCH or its receptor (MCHR1). Using immunocytochemistry and western blotting, we examined whether E₂ decreases MCH-immunoreactive neurones or MCHR1 protein content in the hypothalamus of female rats. We found that both MCH and MCHR1 protein expression was decreased by acute E₂ treatment in ovariectomised rats, and by the peri-ovulatory increase in circulating E₂ in pro-oestrous rats, relative to rats at other cycle stages. To determine whether these changes in MCH/MCHR1 protein expression may be mediated by E₂'s ability to directly regulate the transcription of MCH and MCHR1 genes, the effect of E₂ treatment on MCH and MCHR1 mRNA expression in a neuronal hypothalamic cell line was examined using real-time reverse transcriptase-polymerase chain reaction. We also determined whether MCH and oestrogen receptor (ER)α are co-expressed in the hypothalamus of female rats. E₂ treatment did not decrease MCH or MCHR1 mRNA expression in vitro, and no hypothalamic neurones were identified that co-expressed MCH and ERα. We conclude that E₂-dependent decreases in hypothalamic MCH/MCHR1 protein expression mediate the ability of E₂ to decrease MCH-induced feeding. The current findings suggest, however, that E₂ exerts these actions indirectly, most likely though interactions with other neuronal systems that provide afferent input to MCH and MCHR1 neurones.