The mu-opioid receptor (OR) is involved in several aspects of female reproductive neuroendocrinology, such as the control of gonadotropin release and the display of lordosis behavior. Even though the neuroendocrine events modulated by mu-ORs are steroid hormone-dependent, few studies have shown how steroid hormones such as estrogen and/or progesterone can affect mu-OR function. Therefore, the present study investigated if in vivo estrogen or estrogen plus progesterone treatment of ovariectomized (OVX) rats affects mu-OR coupling to its G proteins. We used autoradiographic analysis of agonist-stimulated [(35)S]-GTPgammaS binding, in which brain sections were incubated in the presence or absence of the mu-OR agonist [D-Ala(2), N-Me-Phe(4), Gly(2)ol]-enkephalin (DAMGO). Film images were quantified using calibrated [(14)C] standards. Analysis was performed in steroid-responsive hypothalamic regions such as the medial preoptic area (mPOA) and the ventromedial nucleus of the hypothalamus, as well as in non-hypothalamic brain regions. Treatment with estrogen, alone or with progesterone, significantly increased DAMGO-stimulated [(35)S]-GTPgammaS binding in the mPOA when compared to control OVX animals. In addition, estrogen increased mu-OR coupling in the caudate putamen. Steroid treatment had no effect on either basal or DAMGO-stimulated binding in the other brain regions examined. These findings suggest that estrogen modulates mu-OR function in a brain region-specific fashion. This could have important implications in terms of how these hormones synchronize reproductive behavior and gonadotropin release.