The xylitol dehydrogenase (EC 1.1.1.9) from xylose-grown cells of Debaryomyces hansenii was partially purified in two chromatographic steps, and characterization studies were carried out in order to investigate the role of the xylitol dehydrogenase-catalyzed step in the regulation of D-xylose metabolism. The enzyme was most active at pH 9.0-9.5, and exhibited a broad polyol specificity. The Michaelis constants for xylitol and NAD(+) were 16.5 and 0.55 mM, respectively. Ca2+, Mg2+, and Mn2+ did not affect the enzyme activity. Conversely, Zn2+, Cd2+, and Co2+ strongly inhibited the enzyme activity. It was concluded that NAD(+)-xylitol dehydrogenase from D. hansenii has similarities with other xylose-fermenting yeasts in respect to optimal pH, substrate specificity, and K-m value for xylitol, and therefore should be named L-iditol:NAD(+)-5-oxidoreductase (EC 1.1.1.14). The reason D. hansenii is a good xylitol producer is not because of its value of K-m for xylitol, which is low enough to assure its fast oxidation by NAD(+)-xylitol dehydrogenase. However, a higher K-m value of xylitol dehydrogenase for NAD(+) compared to the K-m values of other xylose-fermenting yeasts may be responsible for the higher xylitol yields.