Ribitol dehydrogenase (EC 1.1.1.56) of Enterobacter agglomerans strain 221e was purified to homogeneity by polyethylene glycol precipitation, anion exchange chromatography on diethylaminoethyl (DEAE)-Toyopearl 650 M, hydrophobic chromatography on Phenyl-Toyopearl 650 M and gel filtration on Sephadex G-150. The enzyme was specific for NAD(+) but showed a broad substrate specificity for various polyols. Among those tested ribitol exhibited the highest affinity for the enzyme followed by allitol, L-arabitol, L-mannitol, xylitol, L-sorbitol, L-talitol, L-iditol, D-sorbitol, galactitol and erythritol. The enzyme showed maximum activity at pH 9-10 and was stable in the pH range of 9-11. The native enzyme exhibited an isoelectric point of about 4.9 and was estimated to have an apparent M(r) of 92,000 as determined by Sephadex G-150 gelfiltration. SDS-PAGE data suggested that ribitol dehydrogenase of strain 221e has a subunit M(r) of 25,000.