An NAD(+)-dependent L-arabinitol 4-dehydrogenase (LAD, EC 1.1.1.12) from Neurospora crassa was cloned and expressed in Escherichia coli and purified to homogeneity. The enzyme was a homotetramer and contained two Zn2+ ions per subunit, displaying similar characteristics to medium-chain sorbitol dehydrogenases (SDHs). High enzymatic activity was observed for substrates L-arabinitol, adonitol, and xylitol and no activity for D-mannitol, D-arabinitol, or D-sorbitol. The enzyme showed strong preference for NAD(+) but also displayed a very low yet detectable activity with NADP(+). Mutational analysis of residue F59, the single different substrate-binding residue between LADs and D-SDHs, failed to confer the enzyme the ability to accept D-sorbitol as a substrate, suggesting that the amino acids flanking the active site cleft may be responsible for the different activity and affinity patterns between LADs and SDHs. This enzyme should be useful for in vivo and in vitro production of xylitol and ethanol from L-arabinose.