We have hypothesized the importance of a cGMP-dependent, Ca²⁺-activated Cl^- current (I_Cl(cGMP-Ca)) for synchronization of smooth muscle cells (SMCs) in the vascular wall and have demonstrated the dependence of I_Cl(cGMP-Ca) of bestrophin-3 (Best-3) expression. To study the functional role of Best-3, we transfected rat small mesenteric arteries in vivo with siRNA against Best-3 and with non-functional mutated Best-3 siRNA, as a control. The arteries were tested 3 days later in vitro for Best-3 expression, membrane conductance, isometric force development and for intracellular Ca²⁺. Best-3 siRNA significantly reduced mRNA (to 24%) and Best-3 protein (to 35%) expression and abolished I_Cl(cGMP-Ca) without effecting other membrane conductances. Both maximal tension and sensitivity to noradrenalin (NA) were similar in Best-3 siRNA and control arteries. Relaxation to acetylcholine was not affected either. Electrical coupling between SMCs in culture was not changed. The amplitude of NA induced vasomotion was, however, significantly lower (0.08±0.01 vs. 0.17±0.02 N/m; n=11) in arteries with Best-3 siRNA. Vasomotion frequency was not different (0.22±0.01 vs. 0.26±0.03 Hz; n=11) in Best-3 arteries compared to the control. Endothelium denudation abolished vasomotion in both groups. Addition of 8Br-cGMP to the endothelium-denuded vessels restored vasomotion but did not change the difference between groups. SMCs in control endothelium-intact arteries were completely synchronized and had global [Ca²⁺]_i oscillations whereas in the siRNA-transfected arteries only partial synchronization was observe and the asynchronous SMCs had Ca²⁺ waves. Our study demonstrates the importance of Best-3 for synchronization of SMCs and, thus, generation of vasomotion.