Here we have characterized the first transketolase (TK) from a thermophilic microorganism, Geobacillus stearothermophilus, which was expressed from a synthetic gene in Escherichia coli. The G. stearothermophilus TK (mTKgst) retained 100% activity for one week at 50 8C and for 3 days at 658C, and has an optimum temperature range around 60- 70 8C, which will be useful for preparative applications and for future biocatalyst development. The thermostability of the mTKgst allowed us to carry out an easy, one-step purification by heat shock treatment of crude cell extracts at 65 8C for 45 min, directly yielding 132 mg of pure mTKgst from 1 L of culture. The reaction rate of mTKgst with glycolaldehyde was 14 times higher at 70 8C than at 20 8C, and 4 times higher at 50 8C when compared to E. coli TK under identical conditions. When tested at 50 8C with other aldehydes as acceptors, mTKgst activity was approximately 3 times higher than those obtained at 20 8C. Applications of this new TK in biocatalysis were performed with hydroxypyruvate as donor and three different aldehydes as acceptors - glycolaldehyde, d-glyceraldehyde and butyraldehyde - from which the corresponding products l-erythrulose 1, dxylulose 2 and 1,3-dihydroxyhexan-2-one 3 were obtained, respectively. The optical rotations for products 1 and 2 indicate that the stereospecificity of mTKgst is identical to that of other TK sources, leading to a (3S) configuration. With the non-hydroxylated substrate, butanal, the ee value was 85% (3S), showing higher enantioselectivity than the E. coli TK (75% ee, 3S). Processes at elevated temperatures could offer opportunities to extend the applications of TK biocatalysis, by favoring hydrophobic aldehyde acceptor substrate solubility and tolerance towards non-conventional media.