Estradiol mediates structural changes at synapses of the hippocampus, an area in the brain important for learning and memory. This study was designed to test the hypothesis that estradiol mediates subcellular changes of synaptic proteins to induce new synapses via an estrogen receptor (ER)-mediated process. To elucidate the mechanisms involved in glutamatergic synapse formation, we investigated effects of estradiol on synaptic proteins in cultured hippocampal neurons using immunocytochemistry and confocal microscopy. Synaptic protein distribution and size were identified with antibodies to the presynaptic vesicular glutamate transporter protein (vGlut1) and postsynaptic NMDA receptor (NR1 subunit). We observed an increase in synapse density, as detected by NR1 and vGlut1 colocalization, along dendrites of neurons cultured in steroid-stripped media and exposed to estradiol (10 nM) for 48 h. Additionally, the NR1 subunit was enriched at synaptic clusters. Immunocytochemistry and confocal imaging revealed punctate staining of extranuclear ERs along dendrites of hippocampal neurons expressing NR1. Estradiol increased the density of both ER-alpha and ER-beta protein clusters along dendrites. To test whether ERs play an important functional role in the estradiol-induced synaptogenesis, we used the ER antagonist [7alpha,17beta-[9[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI 182,780)] and the ER-alpha- and ER-beta-specific agonists [1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) and 2,3-bis(4-hydroxyphenyl) propionitrile (DPN), respectively]. ICI 182,780 blocked the increase in synapse density. Treatment with PPT, but not DPN, induced significant increases in synapse density that mimicked treatment with estradiol. Together, our results demonstrate that estradiol stimulates glutamatergic synapse formation in the developing hippocampus through an ER-alpha-dependent mechanism. These findings carry profound implications regarding the potential of estrogen to influence learning, memory, and possibly hormone-modulated neurodegeneration.