Objective— Nontranscriptional signaling mechanisms mediate some of the biological effects of estrogen, such as the rapid actions on the blood vessels. By interacting with phosphatidylinositol 3-kinase (PI3K), estrogen receptor (ER) α leads to activation of protein kinase Akt and to subsequent increase in endothelial nitric oxide synthase activity. Because PI3K is mainly a cytoplasmic complex, we studied the cellular site of interaction between this enzyme and ERα, and we dissected the molecular mechanisms that mediate this interaction. Methods and Results— By using cultured human saphenous vain endothelial cells, we found that cell membrane–bound ERα colocalizes with PI3K and may be responsible for PI3K activation. Furthermore, we characterized the subsequent steps in the activation of the PI3K/Akt signaling cascade, comparing the molecular events that follow insulin or estradiol activation of PI3K. Conclusions— We provide novel evidence for an important role of nonnuclear estrogen receptor in rapid, nontranscriptional responses of human endothelial cells to estrogen.