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Molybdenum (Mo) is an essential element for animals, plants, and fungi. To achieve biological activity in eukaryotes, Mo must be complexed into the molybdenum cofactor (Moco). Cells are known to take up Mo in the form of the oxyanion molybdate. However, molybdate transporters are scarcely characterized in the fungal kingdom. In plants and algae, molybdate is imported into the cell via two families of molybdate transporters (MOT), MOT1 and MOT2. For the filamentous fungus Neurospora crassa, a sequence homologous to the MOT1 family was previously annotated. Here we report a characterization of this molybdate-related transporter, encoded by the ncmot-1 gene. We found that the deletion of ncmot-1 leads to an accumulation of total Mo within the mycelium and a roughly 51 % higher tolerance against high molybdate levels when grown on ammonium medium. The localization of a GFP tagged NcMOT-1 was identified among the vacuolar membrane. Thereby, we propose NcMOT-1 as an exporter, transporting molybdate out of the vacuole into the cytoplasm. Lastly, the heterologous expression of NcMOT-1 in Saccharomyces cerevisiae verifies the functionality of this protein as a MOT. Our results open the way towards understanding molybdate transport as part of Mo homeostasis and Moco-biosynthesis in fungi., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)