Dehydroepiandrosterone (DHEA) improves vascular function, but the mechanism of this effect is unclear. Since nitric oxide (NO) regulates vascular function, we hypothesized that DHEA affects the vasculature by increasing endothelial NO production. Physiological concentrations of DHEA stimulated NO release from intact bovine aortic endothelial cells (BAEC) within 5min. This effect was mediated by activation of endothelial nitric oxide synthase (eNOS) in BAEC and human umbilical vein endothelial cells (HUVEC). Dehydroepiandrosterone increased cyclic GMP (cGMP) levels in BAEC, consistent with its effect on NO production. Albumin-conjugated DHEA also stimulated NO release, suggesting that DHEA stimulates eNOS by a plasma membrane-initiated signal. Tamoxifen blocked estrogen-stimulated NO release from BAEC, but did not inhibit the DHEA effect. Pertussis toxin abolished the acute effect of DHEA on NO release. Dehydroepiandrosterone had no effect on intracellular calcium fluxes. However, inhibition of tyrosine kinases or the mitogen-activated protein (MAP) kinase kinase (MEK) blocked NO release and cGMP production in response to DHEA. These findings demonstrate that physiological concentrations of DHEA acutely increase NO release from intact vascular endothelial cells, by a plasma membrane-initiated mechanism. This action of DHEA is mediated by a steroid-specific, G-protein coupled receptor, which activates eNOS in both bovine and human cells. The release of NO is independent of intracellular calcium mobilization, but depends on tyrosine- and MAP kinases. This cellular mechanism may underlie some of the cardiovascular protective effects proposed for DHEA.