Bestrophins are a novel family of proteins that encode calcium-activated chloride channels. In this study we establish that Bestrophin transcripts are expressed in mouse and human heart. Native mBest3 protein expression and localization in heart was demonstrated using a specific polyclonal mBest3 antibody. Immunostaining of isolated cardiac myocytes indicates that mBest3 is expressed at the membrane. Using the patch-clamp technique we characterized the biophysical and pharmacological properties of mBest3 cloned from heart. Whole-cell chloride currents were evoked in both HEK293 and COS-7 cells expressing mBest3 by elevation of intracellular calcium. mBest3 currents displayed a KD for Ca²⁺ of ~175 nM. The calcium-activated chloride current was found to be time- and voltage- independent and displayed slight outward rectification. The anion permeability sequence of the channel was SCN^- > I^- > Cl^- and the current was inhibited by niflumic acid and DIDS in the micromolar range. In addition, we generated a site-specific mutation (F80L) in the putative pore region of mBest3 that significantly altered the ion conduction and pharmacology of this channel. Our functional and mutational studies examining the biophysical properties of mBest3 indicate that it functions as a pore-forming chloride channel that is activated by physiological levels of calcium. This study reports novel findings regarding the molecular expression, tissue localization and functional properties of mBest3 cloned from heart.