The filamentous fungus Aspergillus niger is widely used in biotechnological applications. Strain CBS513.88 is known to harbor 21 copies of the nonautonomous transposon Vader. Upon selection of chlorate-resistant A. niger colonies, one Vader copy was found integrated in the nirA gene. This copy was used for vector construction and development of a transposon-tagging method. Vader showed an excision frequency of about 1 in 2.2 × 10(5) conidiospores. A total of 95 of 97 colonies analyzed exhibited an excision event at the DNA level, and Vader footprints were found. By employing thermal asymmetric interlaced (TAIL)-PCR, the reintegration sites of 21 independent excision events were determined. All reintegration events occurred within or very close to genes. Therefore, this method can be used for transposon mutagenesis in A. niger.