Experiments were conducted to determine if endogenously produced β-endorphin and met-enkephalin exert a physiological inhibition on luteinizing hormone-releasing hormone (LHRH) release in the central nervous system of sheep. Twenty-two mature ewes were implanted with unilateral guide tubes, through which matched infusion cannulae could be inserted without discomfort once daily for intracerebral (i.e.) infusion of three anti-opioid treatments: naloxone (50 µg in 20 µl), sheep antisheep β-endorphin (ABE; 20 µl of 1:25) or sheep anti-met-enkephalin (AME; 20 µl of 1:25) and of two control treatments: nonimmune sheep serum (20 µl of 1:25) or sheep antiporcine thyroglobulin (20 µl of 1:25). To detect abrupt disinhibition of LHRH release by anti-opioid treatments, serum luteinizing hormone (LH) was quantified at 10-min intervals for 1–2 h before and after each i.e. infusion. Complete trials consisted of 3–4 different anti-opioid or control i.e. infusions once daily at a single site over a period of 2–3 days during the luteal phase of recurring estrous cycles. Results were statistically evaluated within each ewe since complete trials were replicated 2–5 times within each ewe and because no 2 ewes could have i.e. infusions in identical locations. Anatomical generalizations were possible when LH responses to anti-opioid treatments were similar for several ewes with i.e. infusion sites in comparable brain regions. However, it was not possible to make such generalizations when infusion sites were not comparable in other ewes. In summary, naloxone consistently disinhibited LHRH/LH when infused into the mediobasal hypothalamus (n = 3), the anterior hypothalamic area (n = 4), the preoptic area (POA; n = 5) and the basal forebrain (BF; n = 5). Intracerebral immunoneutralization of β-endorphin with ABE disinhibited LHRH/LH release when infusions were into the rostral POA/nucleus accumbens region (n = 5) or into the anterolateral hypothalamus (n= 1). Infusions of ABE did not alter LH concentrations when administered into the BF(n = 7) or into areas of the hypothalamus other than the POA (n = 9). Intracerebral immunoneutralization of met-enkephalin with AME was not effective at any ABE-responsive sites (n = 6) or in the BF (n = 5), but it abruptly stimulated LHRH/LH release when infusions were into the anterior hypothalamic area (n = 2) or into the mediobasal hypothalamus (n = 1). Control infusions of antithyroglobulin serum did not alter patterns of serum LH concentrations. In summary, unilateral i.e. immunoneutralization of endogenous β-endorphin and of met-enkephalin in selected brain areas relieved the inhibition of LHRH release in luteal phase ewes. It is concluded that these two endogenous opioid peptides are involved in the physiological regulation of LHRH/LH release in the luteal phase ewe, and that they act at different sites of the centr