Beta-endorphin (beta-END) is an inhibitory factor in the neuroendocrine control of luteinizing hormone (LH) release and thus, presumably also of hypophysiotropic luteinizing hormone-releasing hormone (LHRH) release. In order to address if the median eminence (ME) is a site of beta-END action, we studied its functional role in ewes by assessing: (a) the hypothalamic distribution of beta-END using immunolabeling and by comparing this distribution with our data on the localization of LHRH; (b) the ME in vivo release of LHRH and beta-END during the luteal (day 12) and the follicular (day 15) phases of the estrous cycle; (c) the in vivo release of LHRH from the posterior-lateral ME, as assessed by push-pull cannula (PPC) sampling, before, during, and after infusion of increasing doses of beta-END or naloxone through the PPC, during the follicular phase; and (d) the in vivo release of ME-LHRH and serum LH, before, during, and after infusion of beta-END or naloxone in luteal and follicular ewes. In the ewe, beta-END-containing perikarya are located in and around the arcuate nucleus. Their processes are also present in the diagonal band, medial septal nucleus, and medial and lateral hypothalamic areas, including the preoptic region and posterior ME. Perikarya containing LHRH are located in the preoptic area and project also to the ME, providing opportunities for synaptic interactions between beta-END and LHRH-containing perikarya and processes at these levels. ME in vivo release of LHRH and beta-END increase from the luteal (low LH/high progesterone, P4) to the follicular phase (high LH/low P4). In follicular ewes, in vivo LHRH and LH release is decreased, in a dose-dependent manner, by beta-END infused through the PPC probe into the posterior-lateral ME. In contrast, infusion of naloxone under similar conditions increases LHRH and LH release, also in a dose-dependent fashion. The inhibitory effect of beta-END on LHRH and LH, as well as the stimulatory effect of naloxone on LHRH and LH, were only marginally apparent in luteal ewes. These results suggest that the ME is a major control site where beta-END exerts its influence on hypophysiotropic LHRH release. The strength of this inhibitory effect apparently increases throughout the follicular phase, and might prevent the premature onset of the preovulatory surge of LHRH and LH.