The gene encoding xylose isomerase (xylA) was cloned from Thermus flavus AT62 and the DNA sequence was determined. The xylA gene encodes the enzyme xylose isomerase (XI or xylA) consisting of 387 amino acids (calculated Mr of 44,941). Also, there was a partial xylulose kinase gene that was 4 bp overlapped in the end of XI gene. The XI gene was stably expressed in E. coli under the control of tac promoter. XI produced in E. coli was simply purified by heat treatment at 90 degrees C for 10 min and column chromatography of DEAE-Sephacel. The Mr of the purified enzyme was estimated to be 45 kDa on SDS-polyacrylamide gel electrophoresis. However, Mr of the cloned XI was 185 kDa on native condition, indicating that the XI consists of homomeric tetramer. The enzyme has an optimum temperature at 90 degrees C. Thermostability tests revealed that half life at 85 degrees C was 2 mo and 2 h at 95 degrees C. The optimum pH is around 7.0, close to where by-product formation is minimal. The isomerization yield of the cloned XI was about 55% from glucose, indicating that the yield is higher than those of reported enzymes. The Km values for various sugar substrates were calculated as 106 mM for glucose. Divalent cations such as Mn2+, Co2+, and Mg2+ are required for the enzyme activity and 100 mM EDTA completely inhibited the enzyme activity.