The xylose/glucose isomerase of Saccharococcus caldoxylosilyticus No. 31, a Gram-positive, motile, facultatively anaerobic, thermophilic rod is described in the continuing search for novel enzymes needed to improve industrial production of high fructose corn syrup. The organism was grown in a xylose medium and purified using four stages of chromatography. Yield of enzyme was 17.83% and specific activity, 4.62 U mg-1 protein. Native molecular weight was 160 kDa. Km and Vmax were 111 mM and 2.02 mg min-1 mg-1 protein and 334 mM and 0.92 mg min-1 mg-1 protein, respectively for xylose and glucose substrates at 60°C. Maximal enzyme activity was observed at 60°C. Activation energy was 60.35 kJ mol-1 K-1 and half-life at 70°C was 20 min. This enzyme showed maximal activity at pH 6.4 and stability at pH 7. It required divalent metals, Mn2+>Mg2+>Co2+ for activity and stability. Cu2+ inhibited enzyme activity but Ca2+ did not. The pH profile and non-inhibition by Ca2+ distinguish this enzyme from most glucose isomerases so far characterized.