Three xylose isomerase enzymes (XI) [Eur. J. Biochem. 269 (2002) 157] encoded by mutated xylA genes from Thermus thermophilus were produced at two different levels in Saccharomyces cerevisiae; xylA genes were chromosomally integrated and expressed from multicopy plasmids, respectively. An extra copy of the endogenous xylulokinase gene (XKSI) was chromosomally integrated and the aldose reductase (AR) GRE3 gene was deleted. Ethanol was formed from xylose only when xylA was expressed from multicopy plasmids and when the specific XI activity was higher than 30 mU/mg protein. Deletion of the GRE3 gene was crucial for ethanol formation, possibly because reduced xylitol formation caused less inhibition of XI. (C) 2003 Elsevier Science Inc. All rights reserved.