17Beta-estradiol (E2)-induced neuroprotection is dependent on mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) signaling cascades. We sought to determine whether E2 neuroprotective mechanisms are mediated by a unified signaling cascade activated by estrogen receptor (ER)-PI3K interaction within the same population of neurons or whether E2 activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt are independent signaling events in different neuronal populations. Immunoprecipitation of E2-treated cortical neurons was conducted to determine a protein-protein interaction between ER and the PI3K regulatory subunit p85. Subsequently, cortical neurons were treated with E2 alone or in presence of MAPK inhibitors or PI3K inhibitors. Results of these analyses indicated a protein-protein interaction between ER and p85 that was time-dependent and consistent with the temporal profile for generation of Akt (pAkt) and ERK1/2 phosphorylation (pERK1/2). E2-induced phosphorylation of Akt, was first apparent at 10 min and maximal at 30 min. Simultaneously, E2-induced pERK1/2 was first apparent at 5-10 min and maximal at 30 min. Inhibition of PI3K completely blocked E2 activation of pAkt at 10 and 30 min and blocked E2 activation of ERK1/2 at 10 min, which revealed a PI3K-independent activation of ERK at 30 min. Double immunocytochemical labeling for pERK1/2 and pAkt demonstrated that E2 induced both signaling pathways in the same neurons. These results indicate a unified signaling mechanism for rapid E2 action that leads to the coordinated activation of both pERK1/2 and pAkt in the same population of neurons. Implications of these results for understanding estrogen mechanism of action in neurons and therapeutic development are considered.