Estradiol stimulates endothelial nitric oxide synthase (eNOS) via the activation of plasma membrane (PM)-associated estrogen receptor (ER) α. The process requires Src and erk signaling and eNOS phosphorylation by phosphoinositide 3-kinase (PI3 kinase)-Akt kinase, with Src and PI3 kinase associating with ERα upon ligand activation. To delineate the basis of nongenomic eNOS stimulation, the potential roles of ERα domains necessary for classical nuclear function were investigated in COS-7 cells. In cross-linking studies, estradiol-17β (E₂) caused PM-associated ERα to form dimers. However, eNOS activation by E₂ was unaltered for a dimerization-deficient mutant ERα (ERαL511R). In contrast, ERα mutants lacking the nuclear localization signals (NLS), NLS2,3 (ERαΔ250–274) or the DNA binding domain (ERαΔ185–251), which targeted normally to PM and caveolae/rafts, were incapable of activating eNOS. The loss of NLS2/NLS3 prevented Src and erk activation, and it altered ligand-induced PI3 kinase-ERα interaction and prevented eNOS phosphorylation. Loss of the DNA binding domain did not change E₂ activation of Src or erk, but ligand-induced PI3 kinase-ERα binding and eNOS phosphorylation did not occur. Thus, dimerization is not required for ERα coupling to eNOS; however, NLS2/NLS3 plays a role in Src activation, and the DNA binding region is involved in the dynamic interaction between ERα and PI3 kinase.