The trehalose-synthesizing enzyme, which catalyzes the conversion of maltose to trehalose by intramolecular transglucosylation, was purified from a bacterium, Pseudomonas sp. F1. Its molecular mass was estimated to be 250 kDa by gel filtration and 67 kDa by SDS-polyacrylamide gel electrophoresis, and its pI was 5.8. The native enzyme may consist of 4 subunits. The enzyme was active on maltose and trehalose among saccharides tested as substrates. The N-terminal amino acid of the enzyme was threonine.