17β-Estradiol (E₂) rapidly (<20 min) attenuates the ability of μ-opioids to hyperpolarize guinea pig hypothalamic (β-endorphin) neurons. In the current study, we used intracellular recordings from guinea pig hypothalamic slices to characterize the receptor and intracellular effector system mediating the rapid effects of E₂. E₂ acted stereospecifically with physiologically relevant concentration dependence (EC₅₀ = 8 nm) to cause a 4-fold reduction in the potency of a μ-opioid agonist to activate an inwardly rectifying K⁺conductance. Using Schild analysis to estimate the affinity of the μ-opioid receptor for an antagonist (naloxone), we found that estrogen did not compete for the μ-opioid receptor or alter the affinity of the μ receptor. Both the nonsteroidal estrogen diethylstilbestrol and the “pure” antiestrogen ICI 164,384 blocked the actions of E₂, the latter with a subnanomolar affinity. The protein synthesis inhibitor cycloheximide did not block the estrogenic uncoupling of the μ-opioid receptor from its K⁺ channel, implying a nongenomic mechanism of action by E₂. The actions of E₂ were mimicked by the protein kinase A (PKA) activators forskolin and cAMP, Sp-isomer triethylammonium salt. Furthermore, the selective PKA antagonists cAMP, Rp-isomer triethylammonium salt and KT5720, which have different chemical structures and modes of action, both blocked the effects of E₂. Thus, estrogen binds to a specific receptor that activates PKA to rapidly uncouple the μ-opioid receptor from its K⁺ channel. Because we have previously shown that γ-aminobutyric acid_B receptors are also uncoupled by estrogen, this mechanism of action has the potential to alter synaptic transmission via G protein-coupled receptors throughout the brain.