Estrogen in females protects against cardiovascular disease (CVD) however, hormone replacement therapy (HRT) provides limited protection and has been linked to breast cancer. Dietary phytoestrogens may serve as alternative estrogen receptor (ER) modulators as they bind preferentially to ERb over ERa. The present study characterises intracellular signalling pathways involved in the acute activation of eNOS by phytoestrogens. Human umbilical vein endothelial cells (HUVEC) were treated acutely (0–15 min) with 17b-estradiol (100nM) or the phytoestrogens genistein or equol (100nM). Immunoblotting showed that eNOS-Ser1177 was phosphorylated by equol (2–10min, 100nM) with concurrent phosphorylation of Akt and the mitogen activated protein kinases (MAPK) p38MAPK and p42/44MAPK. Immunoprecpitation experiments demonstrated dissociation of caveolin-1, the negative regulator of eNOS activity, from eNOS with concurrent increased association of HSP90 with eNOS following equol (2 min, 100nM) treatment. The present study establishes that phytoestrogens acutely stimulate NO production in HUVEC via activation of eNOS, Akt, p38MAPK and p42/44MAPK. These results provide the first evidence that phytoestrogens, similar to 17b-estradiol can rapidly phosphorylate eNOS through dissociation of caveolin-1 and association of HSP90. Supported by BBSRC.