We previously reported that dietary isoflavones mediate vascular relaxation via rapid activation of endothelial nitric oxide synthase (eNOS) (Joy et al., 2006) and hypothesized that isoflavone-stimulated production of reactive oxygen species (ROS) may activate induction of antioxidant defense enzymes via the Nrf2/ARE signaling pathway (Siow et al., 2007; Mann et al., 2009). We have now compared the effects of the estrogen receptor (ER-β) agonist DPN with the estrogen receptor alpha agonist PPT (and antagonist ICI 182,780) on eNOS and Nrf2/ARE mediated gene expression in human umbilical vein endothelial cells. Confluent umbilical vein endothelial monolayers were serum-deprived (1% FCS) for 4 h and then treated for 30min with ICI 182,780 (0.01-10 µM) and superoxide production measured using a lucigenin (1 µM) chemiluminescence assay. Serum-deprived endothelial cells (1% FCS for 4 h) were also treated for 3-24 h with vehicle (0.1% DMSO), ICI 182,780 (0.01-10 µM) and cell lysates extracted for SDS-PAGE analysis of heme oxygenase-1 (HO-1), endothelial nitric oxide synthase (eNOS), NAD(P)H:quinone oxidoreductase-1 (NQO1) or glutathione peroxidase-1 (GPx-1) expression. DPN evoked a concentration-dependent increase in superoxide anion production and in HO-1 and eNOS expression, with maximal protein expression detected after 8-12 h. DPN had negligible effects on NQO1 or GPx-1 expression. Similar to the actions of isoflavones, ER antagonist may stimulate intracellular ROS production and kinase pathways leading to Nrf2/ARE mediated gene expression.