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Estrogen receptors (ERs) and estrogen-binding proteins have been localized intracellularly and on the cell surface. The membrane-associated proteins initiate signaling that activates a myriad of cellular responses including the modulation of ion channels and ultimately transcription. Although many of the downstream actions of membrane ERs, including ERα and ERβ, have been characterized, the mechanisms regulating membrane ER levels have remained elusive in the nervous system. In the present study, we used surface biotinylation to identify and study the estradiol regulation of membrane ERα in mixed-sex, cultured hypothalamic neurons from rat. Following surface biotinylation, Western blot analysis revealed full-length 66 kDa ERα and several ERα splice variants, most notably a biotinylated 52 kDa ERα-immunoreactive protein. Treatment of the neurons with estradiol caused a rapid and transient increase of the biotinylated 52 kDa and 66 kDa ERα proteins in the plasma membrane. Exposure of the neurons to estradiol also significantly increased internalization of 52 kDa and 66 kDa ERα membrane proteins, a measure of receptor activation. In the hypothalamus, membrane ERα signaling depends on transactivation of metabotropic glutamate receptor-1a (mGluR1a). Estradiol treatment increased the internalization of mGluR1a in parallel with ERα, a finding consistent with the hypothesis of an ERα-mGluR1a signaling unit. These results demonstrate that estradiol regulates the amount of ERα in the membrane, suggesting estradiol can regulate its own membrane signaling.