A thermostable D-xylose isomerase from a newly isolated thermophilic Streptomyces sp. (PLC) strain is described. The enzyme was purified to homogeneity. It is a homotetramer with a native molecular mass of 183 kDa and a subunit molecular mass of 46 kDa. The enzyme has a K-m of 35 mM for D-xylose and also accepts D-glucose as substrate, however, with a tenfold higher K-m (0.4 M) and half the maximum velocity. Both the activity and stability of this D-xylose isomerase depend strongly on divalent metal ions. Two metal ions bind per subunit to non-identical sites. Mg2+, Mn2+ and Co2+ are of comparable efficiency for the D-xylose isomerase reaction. Co2+ is the most efficient cofactor for D-glucose isomerization. The enzyme remains fully active up to 95 degrees C. The activity decreases at 53 degrees C in the presence of Co2+ and Mg2+ with a half-life of 7 and 9 days respectively. In the presence of Mn2+ the enzyme activity remains constant for at least 10 days and at 70 degrees C 50% of the activity is lost after 5 days.