The fungal transposon Restless of Tolypocladium inflatum ATCC 34921 is a member of the hAT family of mobile DNA elements. In order to study the distribution of this transposon we have looked at 13 fungal strains, most of which are taxonomically related to strain ATCC 34921. Three strains, which show identical banding patterns in a comparative RAPD analysis with strain ATCC 34921, similarly carry multiple copies of Restless. In addition, 1 T. inflatum strain and 2 Beauveria nivea strains contain only a few or even single copies of the transposon. Inverse PCR and DNA sequencing analysis revealed that 1 strain contains a nonmobile truncated version of the element, while the other one harbors a full-length transposon copy which was named Restless-2. The presence of a single transposon copy of Restless in a defined Beauveria strain indicates recent acquisition of this transposon, since class II transposons usually occur in several copies per haploid genome. Notably, the corresponding genomic location is not occupied by a transposon copy in strain ATCC 34921.