Bestrophin-1 is an integral membrane protein encoded by the BEST1 gene and localized predominantly at the basolateral membrane of the retinal pigment epithelium (RPE). Mutations in the BEST1 gene were first associated with Best vitelliforme macular dystrophy (BMD), BMD), a relatively frequent and easily identifiable central retinopathy with autosomal dominant inheritance and variable penetrance. To date, over 120 disease-causing mutations were identified, the majority of which are non-synonymous changes resulting in amino acid substitutions mainly within four mutational hot spot regions in the highly conserved N-terminal half of the protein. While initially thought to impair Cl^- channel function, the molecular pathology of BEST1 mutations still remains controversial. To further study the cellular effects of BMD mutations, we have now analyzed the subcellular localization and anion conductance of 13 disease-associated BEST1 mutations. Subcellular distribution of mutant proteins was examined in polarized MDCK II cells, an established model of apical to basolateral protein sorting. Immunostaing demonstrated a failure of 9 out of 13 mutant proteins to integrate into the cell membrane. Instead, the defective proteins were predominantly retained in the cytoplasm, while wild type bestrophin-1 revealed cell membrane localization. Functional analysis of bestrophin-1 mutants by Iodide fluxes in HEK-293 cells showed that all mutants exhibited significant reduction in anion conductance. Together, our data indicate that a defective intracellular trafficking could be a common cause of BMD accompanied by an impaired anion conductance representing a loss of anion channel function likely due to mistargeting of mutant proteins.