Estrogen plays a role in restoring homeostatic balance during the stress response by altering hypothalamic function and NO production in the brain. While we know that estrogen acts on the hypothalamus to stimulate the NO system through an ERbeta-dependent mechanism in neurons, the molecular mechanisms responsible for these effects are unknown. Because phosphorylation of nNOS at Ser(1412) increases nNOS activity which leads to increased NO production, we investigated the effects of ERbeta activation on nNOS phosphorylation at Ser(1412) and NO production in primary hypothalamic neurons. Using the selective ERbeta agonist, DPN (10nM), we show that activation of ERbeta rapidly increases phosphorylation levels of nNOS at Ser(1412) and NO production. We also show that the PI3K pathway, but not the MAPK pathway, mediates the increases in levels of Ser(1412) phosphorylation and NO production induced by ERbeta activation, as the selective PI3K inhibitor, LY294002 (10microM), blocked the effects of ERbeta activation. Finally, we demonstrate that Src kinase acts upstream of the PI3K/Akt pathway based on our finding that the selective Src inhibitor, PP2 (10microM), blocked the increases in nNOS phosphorylation levels, NO production, and PI3K/Akt activity induced by ERbeta activation. Together, our results show that Src kinase mediates ERbeta-induced increases in phosphorylation levels of nNOS at Ser(1412) and NO production by activating the PI3K/Akt pathway. These findings provide novel insight into the signaling mechanisms through which E2 stimulates the NO system in hypothalamic neurons.