Nanoparticles are very important materials for implementing nanotechnology in diverse areas and are abundant in nature as living organisms operate at a nanoscale. As nanoparticles exhibit interesting size- and shape-dependent physical and chemical properties, the synthesis of uniform nanoparticles with controlled sizes and shapes is of great importance. Nanoparticles are the end products of a wide variety of physical, chemical and biological processes, some of which are novel and radically different and others of which are quite commonplace. The ability to produce nanoparticles with specific shapes and controlled sizes could result in interesting new applications that can potentially be utilized in areas such as optics, electronics and the biomedical field. In the present study, we have demonstrated the ability of the thermophilic bacterium Thermus scotoductus SA-01 to synthesize gold nanoparticles and determined the effect of the physico-chemical parameters on particle synthesis. Furthermore, a protein purified from this bacterium is shown to be capable of reducing HAuCl4 to form elemental nanoparticles in vitro. The protein was purified to homogeneity and identified through N-terminal sequencing as an ABC transporter, peptide-binding protein. It is speculated that this protein reduces Au(III) through an electron shuttle mechanism involving a cysteine disulphide bridge. Through manipulation of physico-chemical parameters, it was possible to vary nanoparticles in terms of number, shape and size. This is the first report of a transporter protein from a thermophile with the ability to produce nanoparticles in vitro thus expanding the limited knowledge around biological gold nanoparticle synthesis.