Genetic improvements of Zymomonas mobilis for pentose utilization have a huge potential in fuel ethanol production. The production of xylitol and the resulting growth inhibition by xylitol phosphate have been considered to be one of the important factors affecting the rates and yields from xylose metabolism by the recombinant Z. mobilis, but the mechanism of xylitol formation is largely unknown. Here, we reported that glucose-fructose oxidoreductase (GFOR), a periplasmic enzyme responsible for sorbitol production, catalyzed the reduction of xylose to xylitol in vitro, operating via a ping-pong mechanism similar to that in the formation of sorbitol. However, the specific activity of GFOR for sorbitol was higher than that for xylitol (68.39 vs. 1.102 micromol min(-1) mg(-1)), and an apparent substrate-induced positive cooperativity occurred during the catalyzed formation of xylitol, with the Hill coefficient being about 2. While a change of the potential acid-base catalyst Tyr269 to Phe almost completely abolished the activity toward xylose as well as fructose, mutant S116D, which has been shown to lose tight cofactor binding, displayed an even slower catalytic process against xylose.