Previous work established that intravenous administration of the opioid receptor antagonist naloxone abruptly increased release of luteinizing hormone (LH) and decreased release of prolactin (PRL) in suckled anestrous ewes and also increased LH release in cyclic luteal ewes. The goal of the present research was to identify brain sites at which local unilateral infusions of naloxone would consistently duplicate the previously noted effects of intravenous naloxone. Intracerebral guide tubes were surgically implanted into the brain of 13 nonpregnant and 16 pregnant ewes at least 4 weeks prior to experimentation. Intracerebral infusion (20–40 µl each through an inner cannula) was performed once daily during postpartum anestrus in suckled fall-lambing ewes and during recurring luteal states of the estrous cycle. Naloxone infusion (n = 142) usually consisted of 50 or 100 µg naloxone, although 5 ewes received 200 and 400 µg per infusion. Control infusions (n = 103) consisted of the vehicle for naloxone (i.e., 0.9% NaCl). Serum concentrations of LH and PRL were quantified at 10-min intervals from 90 min before to 100 min after infusion. Hormone data from individual ewes were grouped for least-squares analysis of variance based upon postmortem neuroanatomical identification of each infusion site. Unilateral intracerebral administration of naloxone consistently induced an increase in LH release within 20 min in the following two neuroanatomical groups: basal forebrain (n = 9 ewes) and chiasmatic area (n = 4 ewes). These naloxone-sensitive brain areas constituted an apparent continuum of tissue sites located as far rostral as ventrolateral septum, diagonal band of Broca and nucleus accumbens and continuing caudally into the preoptic area in and around the organum vasculosum of the lamina terminalis. Brain sites at which the present unilateral infusions of naloxone did not consistently stimulate release of LH included those hypothalamic areas caudal to the preoptic area such as anterior, ventromedial and lateral hypothalamic area including the arcuate nucleus and third ventricle. Therefore, neuroanatomical sites at which local unilateral infusion of naloxone stimulated (i.e., disinhibited) release of LH were very similar to the location of LH-releasing hormone (LHRH) perikarya reported by others for the ovine brain and quite distant from the pituitary gland. Although intravenous naloxone was shown previously to decrease PRL release in suckled anestrous ewes, the present infusion of naloxone did not consistently affect PRL release in such ewes. In summary, local unilateral antagonism of opioid receptors in the vicinity of LHRH perikarya was sufficient to disinhibit release of LHRH/LH, but opioid mechanisms stimulatory to PRL release were not antagonized by intracerebral infusions of nalo