Trehalases play a central role in the metabolism of trehalose and can be found in a wide variety of organisms. A periplasmic trehalase (alpha,alpha-trehalose glucohydrolase, EC 3.2.1.28) from the thermophilic bacterium Rhodothermus marinus was purified and the respective encoding gene was identified, cloned and overexpressed in Escherichia coli. The recombinant trehalase is a monomeric protein with a molecular mass of 59 kDa. Maximum activity was observed at 88 degrees C and pH 6.5. The recombinant trehalase exhibited a K(m) of 0.16 mM and a V(max) of 81 micromol of trehalose (min)(-1) (mg of protein)(-1) at the optimal temperature for growth of R. marinus (65 degrees C) and pH 6.5. The enzyme was highly specific for trehalose and was inhibited by glucose with a K(i) of 7 mM. This is the most thermostable trehalase ever characterized. Moreover, this is the first report on the identification and characterization of a trehalase from a thermophilic bacterium.