Rhythmic contraction, vasomotion, in small rat mesenteric arteries is dependent on a cGMP-mediated interaction between intracellular Ca²⁺ release and membrane potential changes (Peng et al., 2001). We have previously demonstrated the importance of a depolarizing Cl^- conductance for smooth muscle cells (SMCs) synchronization and characterized a cGMP-dependent Ca²⁺ -activated Cl^- current (I_Cl(cGMP-Ca)) in these SMCs. Using a siRNA-based approach we have recently demonstrated that I_Cl(cGMP-Ca) depends on bestrophin-3 (Best-3) expression (Matchkov et al., 2008). In this study we have used the siRNA approach in vivo to highlight the functional significance of Best-3 associated Cl^- current. We transfected rat mesenteric small arteries with Best-3-directed siRNA and with non-functional mutated Best-3 siRNA, as a control. The arteries were studied 3 days later in vitro. A significant reduction (62±6%; n=6) in Best-3 protein was accompanied with inhibition (n=12) of I_Cl(cGMP-Ca) without effects on other membrane conductances. Downregulation of Best-3 did not significantly affect maximal isometric tension and sensitivity to noradrenaline (NA) as well as relaxation to acetylcholine. The amplitude of vasomotion was, however, reduced to 53±4% of the control (n=10) in Best-3 downregulated arteries. Vasomotion frequency was not affected. This effect was endothelium independent since in the absence of endothelium but presence of the membrane permeable 8Br-cGMP vasomotion amplitude was still reduced in the arteries with low expression of Best-3 compared to control vessels. Our study demonstrates that Best-3-associated Ca²⁺ -activated Cl^- current is important for synchronization of SMCs but not for the tonic contraction of the arterial wall. Matchkov, V, Larsen, P, Rojek, A, Golubinskaya, V, Bouzinova, E, Skou Pedersen, F, Aalkjaer, C & Nilsson, H. 2008 Circ Res, 103, 864–872. Peng, H, Matchkov, V, Ivarsen, A, Aalkjaer