The purpose of this study was to determine the role of endogenous opioid peptides in the differentiation of the neuroendocrine brain that leads to estradiol-dependent LH release in female but not in male rats. Newborn intact or gonadectomized rats were given an opiate antagonist, naltrexone, with or without an opiate agonist, morphine, or saline alone during the critical period of the sexual differentiation of the brain (days 1–10). These animals were weaned on day 21 and injected with estradiol benzoate (EB) in oil or oil alone twice, 48 h apart, and the action of EB on plasma and pituitary levels of LH was studied. The release of LH during the prepubertal period was increased following EB treatment both in intact females and gonadectomized males and females, but not in intact male rats. Naltrexone treatment during the neonatal period prevented the EB-induced LH release in both intact and gonadectomized rats. Morphine blocked the inhibitory effect of naltrexone on LH release. Naltrexone treatment did not directly affect pituitary LH storage, but prevented EB-induced depletion of pituitary LH pools; morphine blocked this action of naltrexone. Hence, the inhibitory effect of naltrexone on LH release appeared to result from an alteration of the central mechanism responsible for EB-induced LH secretion. These results suggest a possible involvement of endogenous opioid peptides in the neuroendocrine brain differentiation that results in LH release after EB treatment during the prepubertal period in rat