Prolactin-releasing peptide (PrRP), recently isolated from bovine hypothalamus as an endogenous ligand to a seven transmembrane-domain orphan receptor, is a candidate specific prolactin-releasing factor. The prolactin-releasing activity of the peptide and the expression of the PrRP gene were examined in vivo in relation to estrogen status. Plasma prolactin levels increased significantly with a peak at 5 min after the administration of 50 microg/kg PrRP in female rats in estrus under urethane anesthesia as compared with those in vehicle-treated control rats, but not in female rats in diestrus or proestrus or in male rats. In ovariectomized rats treated with supraphysiological concentration of estrogen, a dose-dependent increase of prolactin secretion in response to 2-50 microg/kg PrRP was observed. However, the peak values induced by 50 microg/kg PrRP were much less than those induced by 2 microg/kg thyrotropin-releasing hormone (TRH). PrRP mRNA levels in the medulla oblongata were decreased by ovariectomy and increased by estrogen treatment. The data indicate that estrogen is prerequisite to the stimulatory effect of PrRP on the secretion of prolactin and to the increase of PrRP mRNA levels in the medulla oblongata. The weak in vivo potency of PrRP on prolactin secretion relative to TRH suggests that PrRP differs from the classical hypophysiotropic hypothalamic releasing hormones.