Trehalose synthase (TreS) catalyzes the reversible interconversion of trehalose (glucosyl-α,α-1,1-glucose) and maltose (glucosyl-α1-4-glucose). TreS was purified from the cytosol of Mycobacterium smegmatis to give a single protein band on SDS gels with a molecular mass of ≈ 68 kDa. However, active enzyme exhibited a molecular mass of ≈ 390 kDa by gel filtration suggesting that TreS is a hexamer of six identical subunits. Based on amino acid compositions of several peptides, the treS gene was identified in the M. smegmatis genome sequence, and was cloned and expressed in active form in Escherichia coli. The recombinant protein was synthesized with a (His)6 tag at the amino terminus. The interconversion of trehalose and maltose by the purified TreS was studied at various concentrations of maltose or trehalose. At a maltose concentration of 0.5 mm, an equilibrium mixture containing equal amounts of trehalose and maltose (42–45% of each) was reached during an incubation of about 6 h, whereas at 2 mm maltose, it took about 22 h to reach the same equilibrium. However, when trehalose was the substrate at either 0.5 or 2 mm, only about 30% of the trehalose was converted to maltose in ≥ 12 h, indicating that maltose is the preferred substrate. These incubations also produced up to 8–10% free glucose. The Km for maltose was ≈ 10 mm, whereas for trehalose it was ≈ 90 mm. While β,β-trehalose, isomaltose (α1,6-glucose disaccharide), kojibiose (α1,2) or cellobiose (β1,4) were not substrates for TreS, nigerose (α1,3-glucose disaccharide) and α,β-trehalose were utilized at 20 and 15%, respectively, as compared to maltose. The enzyme has a pH optimum of about 7 and is inhibited in a competitive manner by Tris buffer. [3H]Trehalose is converted to [3H]maltose even in the presence of a 100-fold or more excess of unlabeled maltose, and [14C]maltose produces [14C]trehalose in excess unlabeled trehalose, suggesting the possibility of separate binding sites for maltose and trehalose. The catalytic mechanism may involve scission of the incoming disaccharide and transfer of a glucose to an enzyme-bound glucose, as [3H]glucose incubated with TreS and either unlabeled maltose or trehalose results in formation of [3H]disaccharide. TreS also catalyzes production of a glucosamine disaccharide from maltose and glucosamine, suggesting that this enzyme may be valuable in carbohydrate synthetic chemistry.