D-Glucose-isomerizing enzyme has been extracted in high yield from D-xylose-grown cells of Bacillus coagulans, strain HN-68, by treating with lysozyme, and purified approximately 60-fold by manganese sulfate treatment, fractionation with ammonium sulfate and chromatography on DEAE-Sephadex column. The purified D-glucose-isomerizing enzyme was homogeneous in polyacrylamide gel electrophoresis and ultracentrifugation and was free from D-glucose-6-phosphate isomerase. Optimum pH and temperature for activity were found to be pH 7.0 and 75°C, respectively. The enzyme required specifically Co++ with suitable concentration for maximal activity being 10-3M. In the presence of Co++, enzyme activity was inhibited strongly by Cu++, Zn++, Ni++, Mn++ or Ca++. At reaction equilibrium, the ratio of D-fructose to D-glucose was approximately 1.0. The enzyme catalyzed the isomerization of D-glucose, D-xylose and D-ribose. Apparent Michaelis constants for D-glucose and D-xylose were 9×10-2M and 7.7×10-2M, respectively.