The trehalose-phosphate synthase (TPS) of Mycobacterium smegmatis was previously purified to apparent homogeneity and several peptides from the 58 kDa protein were sequenced. Based on that sequence information, the gene for TPS was identified in the Mycobacterium tuberculosis genome, and the gene was cloned and expressed in Escherichia coli with a (His)6 tag at the amino terminus. The TPS was expressed in good yield and as active enzyme, and was purified on a metal ion column to give a single band of ≈ 58 kDa on SDS/PAGE. Approximately 1.3 mg of purified TPS were obtained from a 1-L culture of E. coli (≈ 2.3 g cell paste). The purified recombinant enzyme showed a single band of ≈ 58 kDa on SDS/PAGE, but a molecular mass of ≈ 220 kDa by gel filtration, indicating that the active TPS is probably a tetrameric protein. Like the enzyme originally purified from M. smegmatis, the recombinant enzyme is an unusual glycosyltransferase as it can utilize any of the nucleoside diphosphate glucose derivatives as glucosyl donors, i.e. ADP–glucose, CDP–glucose, GDP–glucose, TDP–glucose and UDP–glucose, with ADP–glucose, GDP–glucose and UDP–glucose being the preferred substrates. These studies prove conclusively that the mycobacterial TPS is indeed responsible for catalyzing the synthesis of trehalose-P from any of the nucleoside diphosphate glucose derivatives. Although the original enzyme from M. smegmatis was greatly stimulated in its utilization of UDP–glucose by polyanions such as heparin, the recombinant enzyme was stimulated only modestly by heparin. The Km for UDP–glucose as the glucosyl donor was ≈ 18 mm, and that for GDP–glucose was ≈ 16 mm. The enzyme was specific for glucose-6-P as the glucosyl acceptor, and the Km for this substrate was ≈ 7 mm when UDP–glucose was the glucosyl donor and ≈ 4 mm with GDP–glucose. TPS did not show an absolute requirement for divalent cations, but activity was increased about twofold by 10 mm Mn2+. This recombinant system will be useful for obtaining sufficient amounts of protein for structural studies. TPS should be a valuable target site for chemotherapeutic intervention in tuberculosis.