Pullularia pullulans grown on D-xylose induces the synthesis of a xylitol dehydrogenase capable of oxidizing xylitol to D-xylulose in the presence of NAD according to the following reaction: Xylitol + NAD+ in equilibrium to D-xylulose + NADH + H+. Cells grown on D-glucose do not show appreciably xylitol dehydrogenase activity. However, synthesis of the enzyme begins when those cells were transferred to a liquid medium containing D-xylose. This evidence suggests that xylitol dehydrogenase is an induced enzyme. The purified xylitol dehydrogenase of Pullularia pullulans is quite specific for xylitol, unlike similar dehydrogenases from other sources, which attack a variety of polyols of variable chain length. This property makes the enzyme useful for the determination of xylitol, even in the presence of other polyols.