Two distant homologues of the bestrophin gene family have been identified in the filamentous fungus, Aspergillus nidulans (anbest1 and anbest2). AnBEST1 was functionally characterised using the patch clamp technique and was shown to be an anion selective channel permeable to citrate. Furthermore, AnBEST1 restored the growth of the pdr12Δ yeast mutant on inhibitory concentrations of extracellular propionate, benzoate and sorbate, also consistent with carboxylated organic anion permeation of AnBEST1. Similar to its animal counterparts, AnBEST1 currents were activated by elevated cytosolic Ca2+ with a Kd of 1.60 μM. Single channel currents showed long (>10 s) open and closed times with a unitary conductance of 16.3 pS. Transformation of A. nidulans with GFP-tagged AnBEST1 revealed that AnBEST1 localised to the plasma membrane. An anbest1 null mutant was generated to investigate the possibility that AnBEST1 mediated organic anion efflux across the plasma membrane. Although organic anion efflux was reduced from anbest1 null mutants, this phenotype was linked to the restoration of uracil/uridine-requiring A. nidulans strains to uracil/uridine prototrophy. In conclusion, this study identifies a new family of organic anion-permeable channels in filamentous fungi. We also reveal that uracil/uridine-requiring Aspergillus strains exhibit altered organic anion metabolism which could have implications for the interpretation of physiological studies using auxotrophic Aspergillus strains.