The present study tested the hypotheses that exposure to morphine in utero (10 mg/kg twice a day on gestation days 11–18) and acute estrogen treatment in adulthood alter µ-opioid regulation of hypothalamic norepinephrine (NE) release in sexually mature rats. Both basal and KCl-stimulated NE releases were measured in superfused hypothalamic and preoptic area (POA) slices preloaded with <sup>3</sup>H-NE. The µ-opioid receptor agonist D-Ala<sup>2</sup>, MePhe<sup>4</sup>-Gly-ol<sup>5</sup>-enkephalin (DAMGO; 10 or 100 n M) or the opioid antagonist naloxone (1 µ M) was applied to some slices 15 min prior to KC1 stimulation. Prenatal morphine had no effect on basal or KCl-stimulated release of preloaded <sup>3</sup>H-NE from hypothalamic and POA slices from adult male progeny. Neither the µ-opioid agonist DAMGO nor the nonspecific antagonist naloxone significantly affected KCl-evoked overflow of <sup>3</sup>H-NE in slices from either brain region of male rats. Adult female offspring were ovariectomized (OVX), and some were injected with a replacement dose of estrogen 48 h prior to sacrifice. Prenatal morphine had no effect on basal or KCl-stimulated release of <sup>3</sup>H-NE from hypothalamic or POA slices or on the response of slices to opioid drugs. Estrogen treatment modestly increased KCl-evoked release of <sup>3</sup>H-NE from POA slices from females. Moreover, there was a significant interaction between opioid drugs and estrogen treatment on KCl-evoked overflow of <sup>3</sup>H-NE. In hypothalamic and POA slices from OVX females, DAMGO reduced KCl-evoked efflux of <sup>3</sup>H-NE. This inhibitory effect of DAMGO was not apparent in slices from estrogen-treated females. In addition, naloxone increased KCl-stimulated NE release in slices from both hypothalamus and POA of estrogen-treated female rats but not control OVX females. Thus, prenatal exposure to morphine does not alter basal, KCl-evoked or µ-opioid modulation of NE efflux from hypothalamic or POA slices in adult progeny of either sex. However, treatment of adult, OVX females with estrogen attenuates µ-opioid inhibition and promotes the appearance of naloxone facilitation of KCl-evoked <sup>3</sup>H-NE release from hypothalamic and POA slices.