Bestrophins form Ca2+-activated Cl- channels when they are expressed heterologously. Here we report the functional characterization of murine bestrophin 1 (mBest1). We isolated mBest1 transcript from mouse heart and analyzed the biophysical properties and expression of this channel protein using a tetracycline inducible system. mBest1 expression is localized at the membrane of transfected HEK cells, in agreement with its role as a channel. Whole-cell patch clamp experiments revealed a calcium sensitive, time independent chloride current. mBest1 current displayed slight voltage dependence, exhibited an anion permeability sequence of SCN- > I- > Cl- and was sensitive to DIDS and niflumic acid. Anion replacement studies were also performed on mBest2 and mBest3 and differences were observed in their relative permeability and slope conductance to SCN-. Our study provides the first characterization of the biophysical properties of mBest1 and a framework for the elucidation of the physiological role of bestrophins.