Although the biophysical fingerprints (ion selectivity, voltage-dependence, kinetics, etc) of Ca²⁺-activated Cl⁻ currents are well established, their molecular identity is still controversial. Several molecular candidates have been suggested; however, none of them has been fully accepted. We have recently characterized a cGMP-dependent Ca²⁺-activated Cl⁻ current with unique characteristics in smooth muscle cells. This novel current has been shown to coexist with a “classic” (cGMP-independent) Ca²⁺-activated Cl⁻ current and to have characteristics distinct from those previously known for Ca²⁺-activated Cl⁻ currents. Here, we suggest that a bestrophin, a product of the Best gene family, is responsible for the cGMP-dependent Ca²⁺-activated Cl⁻ current based on similarities between the membrane currents produced by heterologous expressions of bestrophins and the cGMP-dependent Ca²⁺-activated Cl⁻ current. This is supported by similarities in the distribution pattern of the cGMP-dependent Ca²⁺-activated Cl⁻ current and bestrophin-3 (the product of Best-3 gene) expression in different smooth muscle. Furthermore, downregulation of Best-3 gene expression with small interfering RNA both in cultured cells and in vascular smooth muscle cells in vivo was associated with a significant reduction of the cGMP-dependent Ca²⁺-activated Cl⁻ current, whereas the magnitude of the classic Ca²⁺-activated Cl⁻ current was not affected. The majority of previous suggestions that bestrophins are a new Cl⁻ channel family were based on heterologous expression in cell culture studies. Our present results demonstrate that at least 1 family member, bestrophin-3, is essential for a well-defined endogenous Ca²⁺-activated Cl⁻ current in smooth muscles in the intact vascular wall.