A trehalose synthase purified from baker's yeast contained 56-kDa, 102-kDa and 123-kDa polypeptides as its main components. The 102-kDa polypeptide was isolated and shown to be a specific trehalose-6-phosphatase. The trehalose-6-phosphate synthase (Tre6P synthase) activator described by Londesborough and Vuorio [(1991) J. Gen. Microbiol. 137, 323–330] was shown to be phosphoglucoisomerase and to function entirely by generating fructose 6-phosphate. Below 35°C, fructose 6-phosphate is a powerful activator of the Tre6P synthase activity of intact trehalose synthase, especially at physiological phosphate concentration, but does not affect its trehalose-6-phosphatase activity nor the Tre6P synthase activity of truncated trehalose synthase containing truncated versions of the 123-kDa polypeptide. At 50°C, activation by fructose 6-phosphate and inhibition by phosphate are greatly decreased, resulting in an unusually high temperature-dependence for the Tre6P synthase activity at a physiological phosphate concentration (2 mM).