Thermus thermophilus is a model strain to unravel the molecular basis of horizontal gene transfer in hot environments. Previous genetic studies led to the identification of a macromolecular transport machinery mediating DNA uptake in an energy-dependent manner. Here, we have addressed how the transporter is energized. Inspection of the genome sequence revealed four putative transport (AAA) ATPases but only the deletion of one, PilF, led to a transformation defect. PilF is similar to transport ATPases of type IV and type II secretions systems but has a unique N-terminal sequence that carries a triplicated GSPII domain. To characterize PilF biochemically it was produced in Escherichia coli and purified. The recombinant protein displayed NTPase activity with a preference for ATP. Gel filtration analyses combined with dynamic light scattering demonstrated that PilF is monodispersed in solution and forms a complex of 590 ± 30 kDa, indicating a homooligomer of six subunits. It contains a tetracysteine motif, previously shown to bind Zn(2+) in related NTPases. Using atomic absorption spectroscopy, indeed Zn(2+) was detected in the enzyme, but in contrast to all known zinc-binding traffic NTPases only one zinc atom was bound to the hexamer. Deletion of the four cysteine residues led to a loss of Zn(2+). Nevertheless, the mutant protein retained ATPase activity and hexameric complex formation.