Bestrophin 3 (Best3) is a calcium-activated chloride channel expressed in heart and other tissues. We hypothesized that Best3 channels function in a macromolecule complex similar to other cardiac ion channels. Yeast two-hybrid assays performed on a mouse heart library, identified histidine-rich calcium binding protein (Hrc) as a novel binding partner of Best3. Hrc is a regulator of sarcoplasmic reticulum (SR) calcium uptake, storage and release. This interaction was further determined to involve Best3 amino acids 541-669 in the cytosolic C-terminus of the channel and the cysteine-rich region in mouse Hrc (amino acids 665-738 aa). Specific binding between Best3 channels and Hrc was verified by co-immunoprecipitation using mouse heart homogenates. Confocal immunofluorescence microscopy further demonstrated a strong co-localization of Best3 and Hrc in isolated ventricular and atrial myocytes. In this study we report that cardiac Best3 channels interact with Hrc. Calcium-activated chloride channels play a critical role in excitation-contraction coupling in the SR by balancing charge movement during calcium release and reuptake. The direct coupling of Best3 with Hrc suggests that Best3 channels may play a role in calcium handling in the heart by functioning as a counterion channel.