BACKGROUND AND AIMS: The aim of the present study was to investigate the effects of prolonged, intermittent infusion of β-endorphin or naloxone into the third cerebral ventricle of follicular phase ewes on kisspeptin (kiss 1) mRNA and RFamide-related peptide-3 (RFRP-3) mRNA levels in the hypothalamus. It was also examined the influence of β-endorphinergic stimulation or blockade on the expression of gonadotropin releasing hormone (GnRH) and GnRH receptor (GnRHR) proteins in the hypothalamic-pituitary unit and on luteinizing hormone (LH) secretion from the anterior pituitary gland. METHODS: The levels of GnRH and GnRHR proteins were analyzed using an enzyme-linked immunoabsorbent assay (ELISA) in selected tissue of the preoptic area-hypothalamic region: preoptic area (POA), anterior hypothalamus (AH), ventromedial hypothalamus (VM), stalk/median eminence (SME), and GnRHR in the anterior pituitary gland (AP). The Real-time PCR with SYBR Green dye was used for evaluation of kiss 1 mRNA in the POA and arcuate nucleus (ARC) and RFRP3 mRNA in the paraventricular nucleus (PVN) and in the dorsomedial hypothalamus (DMH). RESULTS: Stimulation of β-endorphin receptors significantly decreased the levels of GnRH protein and kiss 1 transcript in all analyzed structures and usually led to similar responses in the expression of GnRHR. Precisely, β-endorphin decreased the level of GnRHR protein in the POA, MBH, SME and AP, but had no significant influence on the receptor quantity in the AH. In addition, β-endorphin decreased LH secretion. Naloxone had an opposite effect on proteins biosynthetic level. CONCLUSIONS: The obtained results suggest that β-endorphin can modulate the biosynthesis and release of GnRH through complex changes in the expression of kiss 1 mRNA and GnRHR protein in the hypothalamus. It also appears, that in sheep β-endorphin influences GnRH/LH secretion by mechanism(s) excluding RFRP-3 neuronal system.