The risk of cardiovascular disease is increased in postmenopausal women and growing concerns over the use of hormone replacement therapy (HRT) have prompted a search for alternative therapy. We have shown that dietary phytoestrogens (genistein, daidzein and its metabolite equol) modulate endothelium dependent relaxation in vitro and lower blood pressure in vivo (Mahn et al., 2005). More recently, we reported that physiological concentrations of equol (100nM) rapidly (2min) activate eNOS via phosphorylation of Akt and ERK1/2 (Joy et al., 2006). In the present study, we have investigated whether equol (100nM) induced eNOS activation is associated with (i) increased production of reactive oxygen species (ROS), (ii) eNOS dissociation from caveolin-1 and (iii) changes in the F-actin cytoskeleton. Human umbilical vein endothelial cells (HUVEC) were incubated in Krebs Henseleit buffer containing lucigenin (5µM), vehicle (DMSO) or equol (100nM) in the presence or absence of NADPH oxidase inhibitors apocynin (10µM) and diphenylionium (1µM) or the mitochondrial complex-I inhibitor rotenone (5µM). HUVEC were also serum deprived for 4h before treatment (2min) with vehicle (DMSO) or equol (100nM) and confocal immunofluorescent staining for eNOS (475_ex 515_em). Mitochondrial superoxide production was detected in serum deprived HUVEC loaded with Mitosox dye for 30min. Cells were then treated for 20min with vehicle (DMSO), equol (100nM) or the mitochondrial complex-III inhibitor antimycin-A (100ng ml^-1). Similarly, Phalloidin was used to visualize F-actin by confocal fluorescence microscopy using wavelengths (nm) 560_ex 625_em for Mitosox or Phalloidin, and 375_ex 450_em for nuclear Hoechst staining. Inhibition of NADPH oxidase and mitochondrial complex I abrogated equol stimulated ERK1/2, Akt and eNOS phosphorylation (n=4 cultures, p<0.05 Student’s t-test). Equol increased mitochondrial superoxide production, with basal fluorescence (96 ± 5 arbitrary units, mean ± SEM, n=4 cultures) elevated by ~50% (141 ± 7, n=4 cultures, p<0.01 Student’s t-test) and stimulated ROS production was attenuated by inhibitors of NADPH oxidase and rotenone (n=4 cultures, p<0.05 Student’s t-test). Immunostaining for F-actin and eNOS revealed alterations in their intracellular distribution. eNOS was found to translocate from the membrane to the cytosol and was accompanied by the appearance of F-actin stress fibers, highlighting that equol may alter intracellular eNOS trafficking. In summary, equol stimulated mitochondrial ROS production may be important for activation of eNOS, with equol induced alterations in the F-actin cytoskeleton modulating mitochondrial ROS production (Felty et al., 2005) and subsequent eNOS activation.