Oestrogen protects premenopausal women against heart disease; however, hormone replacement therapy following menopause has been linked to breast cancer and has limited benefits on the cardiovascular system (Rossouw et al. 2002). Dietary phytoestrogens, such as genistein and the isoflavone metabolite equol, may serve as alternative oestrogen receptor modulators that prevent vascular endothelial dysfunction through enhanced generation of the vasodilator nitric oxide (NO; Kuiper et al. 1998; Mahn et al. 2005). The present study investigates whether genistein or equol can acutely modulate intracellular signalling pathways involved in the activation of endothelial NO synthase (eNOS). Confluent cultures of human umbilical vein endothelial cells (HUVEC) were treated acutely (0-10 min) with either genistein or equol (100nM) in Krebs Henseleit buffer containing L-arginine (100 μM) and lysates collected for Western blot analyses. The statistical signigficance of quantified data derived from n=3-6 independent HUVEC cultures were evaluated using Student’s unpaired t tests. Immunoblotting revealed that eNOS-Ser¹¹⁷⁷ was phosphorylated by equol at 2-10 min with concurrent phosphorylation of Akt and extracellular signal-regulated kinase (ERK1/2). Dissociation of caveolin-1 from eNOS and increased eNOS association with HSP90 following equol treatments was demonstrated by eNOS immunoprecipitation. Inhibition of phosphoinositol-3-kinase/Akt using LY294002 (10 μM, 30 min) or MEK1/2 using or U0126 (1 μM, 30 min) resulted in a significant attenuation of eNOS phosphorylation by equol (2 min, 100 nM, p<0.05, n=3). Our present study provides the first direct evidence that isoflavones can acutely phosphorylate eNOS in HUVEC via Akt and ERK1/2 activation to enhance eNOS dissociation from caveolin-1 and association with HSP90. These acute intracellular signalling events mediate the enhanced NO generation and vascular relaxation elicited by isoflavones.