ERM (Ezrin, Radixin & Moesin) proteins link the actin cytoskeleton to membrane proteins either directly or throught EBP50 (NHERF1). Their activation and function in vascular smooth muscle remain to be elucidated. We have investigated the activation of these proteins in intact Wistar rat aorta by using a phospho-ERM antibody and Western-Blot. The role of moesin and EBP50 in the regulation of vascular tone was investigated in mesenteric arteries after inhibition of moesin and EBP50 expression by siRNA transfection. Potential partners of EBP50 were screened by LC/MS-MS analysis of gel bands obtained after co-immunoprecipitation with EBP50. Stimulation with noradrenaline (NA) 1μM rapidly increased ERM phosphorylation, which was maximum at 2 min and slightly decreased at 10 min. ERM proteins were also phosphorylated by 100 mM KCl stimulation with a maximal phosphorylation after 30 s and a fast decrease after longer stimulation. The inhibitor of Rho kinase (Y27632 10 μM), totally inhibited ERM phosphorylation measured after 2 and 10 min of NA stimulation but did not affect the rapid increase (30 s) in ERM phosphorylation induced either by NA or KCl. The rapid increase in ERM phosphorylation induced by KCl stimulation was totally inhibited by nimodipine, a blocker of voltage-gated calcium channels. Furthermore, ionomycin, a calcium ionophore, induced a rapid increase in ERM phosphorylation. Moreover, GÖ6983, an inhibitor of calcium-activated PKC, totally inhibited the fast ERM phosphorylation evoked either by NA or KCl stimulation but not the phosphorylation induced by a longer stimulation with NA. Knock-down of moesin potentiated the noradrenaline-evoked contraction but not the KCl-evoked contraction. A similar effect was observed in EBP50 knock-down arteries, indicating that moesin could exert its effect through an interaction with EBP50. Immunoprecipitation of EBP50 in noradrenaline-stimulated arteries allowed to identify its interaction with moesin and several other proteins involved in cytoskeleton regulation, especially in focal adhesion formation, that did not appear in unstimulated arteries. These results indicate that ERM proteins are phosphorylated in vascular smooth muscle in response to agonist or depolarisation stimulation. This phosphorylation consists in a fast calcium dependent phosphorylation induced by PKC and a much slower calcium independent phosphorylation induced by Rho kinase. Moesin activation is associated with decreased NA-evoked contraction, probably throught interaction with EBP50 and cytoskeleton elements.