Bestrophin-1 (Best1) is a Cl^- channel that is linked to various retinopathies in both humans and dogs. Dysfunction of the Best1 Cl^- channel has been proposed to cause retinopathy because of altered Cl^- transport across the retinal pigment epithelium (RPE). In addition to Cl^-, many Cl^- channels also transport HCO₃. Because HCO₃^- is physiologically important in pH regulation and in fluid and ion transport across the RPE, we measured the permeability and conductance of bestrophins to HCO₃^- relative to Cl^-. Four human bestrophin homologues (hBest1, hBest2, hBest3 and hBest4) and mouse Best2 (mBest2) were expressed in HEK cells and the relative HCO₃^- permeability (P_HCO3/P_Cl) and conductance (G_HCO3/G_Cl) were determined. P_HCO3/P_Cl^- was calculated from the change in reversal potential (E_rev) produced by replacing extracellular Cl^- with HCO₃^-. hBest1 was highly permeable to HCO₃^- (P_HCO3/P_Cl^- = ~0.44). hBest2, hBest4 and mBest2 had an even higher relative HCO₃^- permeability (P_HCO3/P_Cl^- = 0.6 - 0.7). All four bestrophins had HCO₃^- conductances that were nearly the same as Cl^- (G_HCO3/G_Cl^- = 0.9 - 1.1). Extracellular Na did not affect the permeation of hBest1 to HCO₃^-. At physiological HCO₃^- concentration, HCO₃- was also highly conductive. The hBest1 disease-causing mutations Y85H, R92C and W93C abolished both Cl^- and HCO₃^- currents equally. V78C mutation changed P_HCO3/P_Cl^- and G_HCO3/G_Cl^- of mBest2 channels. These results raise the possibility that disease-causing mutations in hBest1 produce disease by altering HCO₃- homeostasis as well as Cl^- transport in the retina.