Dehydroepiandrosterone (DHEA) and its sulfate ester (DHEAS) are the major circulating steroid hormones in humans, and their levels progressively decline with age. Epidemiological studies suggest that DHEA/DHEAS concentrations may be inversely related to cardiovascular risk, but disagreement exists on this issue. Preliminary studies show that DHEA regulates vascular function, but little data has been published on the mechanisms. We show that DHEA administration to human endothelial cells triggers nitric oxide (NO) synthesis, due to enhanced expression and stabilization of endothelial nitric oxide synthase (eNOS). Additionally, DHEA rapidly activates eNOS, through a non-transcriptional mechanism which depends on ERK 1/2 mitogen-activated protein kinases (MAPK) but not on phosphatidylinositol 3-OH kinase/Akt. DHEA is not converted to estrogens nor to androgens by endothelial cells, and its genomic and nongenomic effects are not blocked by antagonists of the estrogen, progesterone, glucocorticoid or androgen receptors, suggesting that DHEA acts through a specific receptor. Oral DHEA administration to ovariectomized Wistar rats dose-dependently restores aortic eNOS amount and eNOS activity, confirming DHEA effects in vivo. Our present data suggest that DHEA may have direct genomic and nongenomic effects on the vascular wall, which are not mediated by other steroid hormone receptors, leading to eNOS activation and induction.