A mutant ribitol dehydrogenase (RDH-F) was purified from Klebsiella aerogenes strain F which evolved from the wild-type strain A under selective pressure to improve growth on xylitol, a poor substrate used as sole carbon source. The ratio of activities on xylitol (500 mM) and ribitol (50 mM) was 0.154 for RDH-F compared to 0.033 for the wild-type (RDH-A) enzyme. The complete amino acid sequence of RDH-F showed the mutations. Q60 for E60 and V215 for L215 in the single polypeptide chain of 249 amino acid residues. Structural modeling based on homologies with two other microbial dehydrogenases suggests that E60 --> Q60 is a neutral mutation, since it lies in a region far from the catalytic site and should not cause structural perturbations. In contrast, L215 --> V215 lies in variable region II and would shift a loop that interacts with the NADH cofactor. Another improved ribitol dehydrogenase, RDH-D, contains an A196 --> P196 mutation that would disrupt a surface alpha-helix in region II. Hence conformational changes in this region appear to be responsible for the improved xylitol specificity.