Trehalose analogues bearing fluorescent and click chemistry tags have been developed as probes of bacterial trehalose metabolism, but these tools have limitations with respect to in vivo imaging applications. Here, we report the radiosynthesis of the 18 F-modified trehalose analogue 2-deoxy-2-[ 18 F]fluoro-D-trehalose ([ 18 F]-2-FDTre), which in principle can be used in conjunction with positron emission tomography (PET) imaging to allow in vivo imaging of trehalose metabolism in various contexts. A chemoenzymatic method employing the thermophilic TreT enzyme from Thermoproteus tenax was used to rapidly (15-20 min), efficiently (70% radiochemical yield; ≥ 95% radiochemical purity), and reproducibly convert the commercially available radiotracer 2deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F]-2-FDG) into the target radioprobe [ 18 F]-2-FDTre in a single step; both manual and automated syntheses were performed with similar results. Cellular uptake experiments showed that radiosynthetic [ 18 F]-2-FDTre was metabolized by Mycobacterium smegmatis but not by various mammalian cell lines, pointing to the potential future use of this radioprobe for selective PET imaging of infections caused by trehalose-metabolizing bacterial pathogens such as M. tuberculosis.