Neurospora crassa X1 was found to ferment xylose and glucose simultaneously. Xylose was the appropriate inducer for the production of xylose reductase that had two isoenzymes designated as EI and EII. Both EI and EII, which were purified by affinity chromatography, had NADPH-dependent xylose reductase activities. EII also had NADH-dependent activity, and EI is the only xylose reductase found so far without any NADH-dependent activity. EI and EII had MWs of 30 kDa and 27 kDa, and pls of 5.6 and 5.2, respectively. The specifities of EI and EII against triose, pentoses, and hexoses were studied. The K(m)s against xylose for EI and EII were 2.3 mM and 1.1 mM respectively, which were much lower than those of the xylose reductase from yeast.