Rhizomucor pusillus NBRC 4578 efficiently produces ethanol from lignocellulosic biomass because of its ability to ferment not only D-glucose, but also D-xylose. When the strain was cultivated on D-xylose, ethanol was gradually formed in the culture medium with a decrease in D-xylose and the simultaneous accumulation of xylitol, which suggested that the strain catabolized D-xylose with D-xylose reductase (XR) and xylitol dehydrogenase (XDH). XR (RpXR) was purified to homogeneity from the crude extract prepared from the mycelia of the strain grown on D-xylose. The purified enzyme was found to be NADPH-dependent and prefer pentoses such as D-xylose, D-ribose, and L-arabinose as substrates. Isolation of the genomic DNA and cDNA of the xyl1 gene encoding RpXR revealed that the gene was interrupted by two introns and the exon of the gene encoded a protein composed of 322 amino acids with a M-r of 36,724. Phylogenetic analysis showed that RpXR is more related to 4-dihydromethyltrisporate dehydrogenases from Mucoraseae fungi rather than the previously reported fungal XRs. Quantitative real-time PCR indicated that transcription of the xyl1 gene was marked in the presence of D-xylose and L-arabinose, but was week in the presence of D-glucose. These biochemical and expression analyses suggest that RpXR is involved in the catabolism of L-arabinose as well as D-xylose. This is the first report of the purification, characterization, and gene cloning of XR from zygomycetous fungi. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.