Dehydroepiandrosterone (DHEA) activates a plasma membrane receptor on vascular endothelial cells and phosphorylates extracellular signal-regulated kinase (ERK) 1/2. We hypothesize that ERK1/2-dependent vascular endothelial proliferation underlies part of the beneficial vascular effect of DHEA. DHEA (0.1–10 nM) activated ERK1/2 in bovine aortic endothelial cells (BAEC) by 15 minutes, causing nuclear translocation of phosphorylated ERK1/2 and phosphorylation of nuclear p90 ribosomal S6 kinase. ERK1/2 phosphorylation was dependent on plasma membrane-initiated activation of Gi/o proteins and on the upstream mitogen-activated protein kinase kinase, since the effect was seen with albumin-conjugated DHEA and was blocked by pertussis toxin or PD098059. A 15-minute incubation of BAEC with 1nM DHEA (or albumin-conjugated DHEA) increased endothelial proliferation by 30% at 24 hours. This effect was not altered by inhibition of estrogen or androgen receptors or by inhibition of nitric oxide production. There was a similar effect of DHEA to increase endothelial migration. DHEA also increased the formation of primitive capillary tubes of BAEC in vitro in solubilized basement membrane. These rapid DHEA-induced effects were reversed by the inhibition of either Gi/o-proteins or ERK1/2. Additionally, DHEA enhanced angiogenesis in vivo in a chick embryo chorioallantoic membrane assay. These findings indicate that exposure to DHEA, at concentrations found in human blood, causes vascular endothelial proliferation by a plasma membrane-initiated activity that is Gi/o- and ERK1/2-dependent. These data, along with previous findings, define an important vascular endothelial cell signaling pathway that is activated by DHEA, and suggest that this steroid may play a role in vascular function.