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 homologs (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. The 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.