This study demonstrates that the estrogen receptor alpha (ERα) interacts with the regulatory subunit p85α of phosphatidylinositol-3-OH kinase (PI(3)K) in a ligand-dependent manner. Estrogen stimulation increases ERα-associated PI(3)K activity, leading to the activation of protein kinase B/Akt and endothelial nitric oxide synthase (eNOS). These effects are independent of gene transcription and do not involve phosphotyrosine adapter molecules or src-homology domains of p85α. The findings show that estrogen's vascular protective effects are mediated through this non-nuclear signaling pathway involving direct ERα-PI(3)K interaction. In mice, estrogen treatment increased eNOS activity and decreased vascular leukocyte accumulation after ischaemia and reperfusion injury, effects that were abolished by PI(3)K or eNOS inhibitors. PI(3)K is involved in various cellular functions, including those of platelet-derived growth factor (PDGF), insulin, and vascular endothelial growth factor (VEGF). The predominant form of PI(3)K consists of p85α and p110. PI(3)K catalyzes the formation of lipid mediators that recruit signaling molecules, including Akt. Akt activation mediates many downstream effects of PI(3)K, including eNOS activation. The study shows that estrogen stimulates eNOS activity through PI(3)K, which is blocked by PI(3)K inhibitors. ERα interacts with p85α in a ligand-dependent manner, and this interaction is not mediated by adapter molecules or src-homology domains. ERα also interacts with other steroid hormone receptors, suggesting a broader role for ERα in non-nuclear signaling. The study also shows that ERα interacts with p85α in a cell-free system and that this interaction does not involve the src-homology SH2/SH3 domains of p85α. Heat shock protein 90 inhibits the interaction of ERα and p85α. The study further shows that estrogen stimulates Akt kinase activity, which is involved in eNOS activation. Transfection experiments with constitutively active or dominant-negative Akt mutants confirm that Akt mediates eNOS activation. The study also shows that estrogen's vascular protective effects are mediated by PI(3)K and NO. The findings suggest that ERα is involved in a critical function outside the nucleus, and that PI(3)K is a key mediator of estrogen's biological effects. The study also shows that ERα is present in membrane and cytoplasmic compartments, suggesting that membrane-associated ER is involved in mediating NO release from endothelial cells. The study provides evidence that ERα can recruit and activate PI(3)K through a subset of ligand-bound, membrane-associated ERs.This study demonstrates that the estrogen receptor alpha (ERα) interacts with the regulatory subunit p85α of phosphatidylinositol-3-OH kinase (PI(3)K) in a ligand-dependent manner. Estrogen stimulation increases ERα-associated PI(3)K activity, leading to the activation of protein kinase B/Akt and endothelial nitric oxide synthase (eNOS). These effects are independent of gene transcription and do not involve phosphotyrosine adapter molecules or src-homology domains of p85α. The findings show that estrogen's vascular protective effects are mediated through this non-nuclear signaling pathway involving direct ERα-PI(3)K interaction. In mice, estrogen treatment increased eNOS activity and decreased vascular leukocyte accumulation after ischaemia and reperfusion injury, effects that were abolished by PI(3)K or eNOS inhibitors. PI(3)K is involved in various cellular functions, including those of platelet-derived growth factor (PDGF), insulin, and vascular endothelial growth factor (VEGF). The predominant form of PI(3)K consists of p85α and p110. PI(3)K catalyzes the formation of lipid mediators that recruit signaling molecules, including Akt. Akt activation mediates many downstream effects of PI(3)K, including eNOS activation. The study shows that estrogen stimulates eNOS activity through PI(3)K, which is blocked by PI(3)K inhibitors. ERα interacts with p85α in a ligand-dependent manner, and this interaction is not mediated by adapter molecules or src-homology domains. ERα also interacts with other steroid hormone receptors, suggesting a broader role for ERα in non-nuclear signaling. The study also shows that ERα interacts with p85α in a cell-free system and that this interaction does not involve the src-homology SH2/SH3 domains of p85α. Heat shock protein 90 inhibits the interaction of ERα and p85α. The study further shows that estrogen stimulates Akt kinase activity, which is involved in eNOS activation. Transfection experiments with constitutively active or dominant-negative Akt mutants confirm that Akt mediates eNOS activation. The study also shows that estrogen's vascular protective effects are mediated by PI(3)K and NO. The findings suggest that ERα is involved in a critical function outside the nucleus, and that PI(3)K is a key mediator of estrogen's biological effects. The study also shows that ERα is present in membrane and cytoplasmic compartments, suggesting that membrane-associated ER is involved in mediating NO release from endothelial cells. The study provides evidence that ERα can recruit and activate PI(3)K through a subset of ligand-bound, membrane-associated ERs.