An NAD(+)-dependent D-arabinitol dehydrogenase (polyol dehydrogenase) gene was isolated from Pichia stipitis CBS 6054 and cloned in Saccharomyces cerevisiae. The gene was isolated by screening of a lambda-cDNA library with a zymogram technique. D-Arabinitol, xylitol, D-glucitol and galactitol are substrates for the recombinant protein. With D-arabinitol as substrate the reaction product is D-ribulose. The molecular weight of the native tetramer enzyme is 110,000 Da and the monomer is 30,000 Da. The amino acid sequence is homologous to the short-chain dehydrogenase family. It is 85.5% identical to a D-arabinitol dehydrogenase from Candida albicans. The gene in P. stipitis was induced by D-arabinitol and P. stipitis was able to grow on D-arabinitol. The physiological role of D-arabinitol metabolism is discussed.