Superoxide anions (O-2(.-)) induce oxidative stress and reduce endothelial NO availability by peroxynitrite formation. In human endothelial cells gp91(phox) was identified as the limiting subunit of the forming NAD(P)H oxidase, Because endothelin-1 (ET-1) is considered as a pro-atherosclerotic stimulus, we analyzed the effect of ET-1 on gp91(phox) expression and O-2(.-) generation in primary cultures of human umbilical vein endothelial cells (HUVECs), The gp91(phox) mRNA expression was quantified by standard calibrated competitive reverse transcriptase-polymerase chain reaction. ET-1 induces gp91(phox) mRNA expression in HUVEC (max, after 1 h), The induction of gp91(phox) expression was dose-dependent, reaching its maximum at 10 nmol/L ET-I, The increased gp91(phox) expression is mediated by endothelial receptor type B (ETB). Furthermore, ET-1 augments O-2(.-) generation in human endothelial cells as measured by coelenterazine chemiluminescence. These data support a new mechanism: how ET-1 increases oxidative stress in the vessel wall leading to endothelial dysfunction and enhanced susceptibility to atherosclerosis.