L-Arabinitol 4-dehydrogenase (Lad1) of the cellulolytic and hemicellulolytic fungus Hypocrea jecorina (anamorph: Trichoderma reesei) has been implicated in the catabolism of L-arabinose, and genetic evidence also shows that it is involved in the catabolism of D-xylose in xylitol dehydrogenase (xdh1) mutants and of D-galactose in galactokinase (gal1) mutants of H. jecorina. In order to identify the substrate specificity of Lad1, we have recombinantly produced the enzyme in Escherichia coli and purified it to physical homogeneity. The resulting enzyme preparation catalyzed the oxidation of pentitols (L-arabinitol) and hexitols (D-allitol, D-sorbitol, L-iditol, L-mannitol) to the same corresponding ketoses as mammalian sorbitol dehydrogenase (SDH), albeit with different catalytic efficacies, showing highest k(cat)/K(m) for L-arabinitol. However, it oxidized galactitol and D-talitol at C4 exclusively, yielding L-xylo-3-hexulose and D-arabino-3-hexulose, respectively. Phylogenetic analysis of Lad1 showed that it is a member of a terminal clade of putative fungal arabinitol dehydrogenase orthologues which separated during evolution of SDHs. Juxtapositioning of the Lad1 3D structure over that of SDH revealed major amino acid exchanges at topologies flanking the binding pocket for d-sorbitol. A lad1 gene disruptant was almost unable to grow on L-arabinose, grew extremely weakly on L-arabinitol, D-talitol and galactitol, showed reduced growth on D-sorbitol and D-galactose and a slightly reduced growth on D-glucose. The weak growth on L-arabinitol was completely eliminated in a mutant in which the xdh1 gene had also been disrupted. These data show not only that Lad1 is indeed essential for the catabolism of L-arabinose, but also that it constitutes an essential step in the catabolism of several hexoses; this emphasizes the importance of such reductive pathways of catabolism in fungi.