ProThyrotropin-releasing hormone (proTRH) is a prohormone widely distributed in many areas of the brain. After biosynthesis, proTRH is subjected to post-translational processing to generate TRH and seven non-TRH peptides. Among these non-TRH sequences, we found previously that preproTRH178-199 could regulate the secretion of prolactin in suckled rats by their pups. Dopamine (DA), the main regulator of prolactin secretion, is produced in dopaminergic tyrosine hydroxylase (TH)-positive neurons in the hypothalamic arcuate nucleus (ARC). In this study we investigated whether prolactin release during the estrous sexual cycle is regulated by preproTRH178-199 through its effect on DA neurons of the ARC. We observed that biotinylated preproTRH178-199 bound to neurons in the ARC; this was higher during proestrus than during diestrus. Binding of preproTRH178-199 to DA neurons was seen only during proestrus in the ARC. Using primary neuronal hypothalamic cultures we found that preproTRH178-199 peptide decreased TH levels in a dose-responsive manner, whereas intra-ARC administration of preproTRH178-199 induced a 20-fold increase in plasma prolactin levels. Together, these results suggest a potential role for preproTRH178-199 in regulating dopaminergic neurons involved in the inhibition of pituitary prolactin release.