An alcohol dehydrogenase produced by Burkholderia sp. AIU 652, which was isolated with a cholesterol medium, was purified to homogeneity and characterized. The enzyme had broad substrate specificity, and the best reaction was the reversible oxidation of 2-propanol to acetone and 2-butanol to 2-butanone. The K-m values for secondary alcohols and ketones were much lower than those for primary alcohols or diols. In addition, the enzyme oxidized R-(-)-alcohols in preference to S-(+)-alcohols, and utilized NAD(+), but not NADP(+) as the cofactor. The molecular mass was 150 kDa with four identical subunits, and the activity was inhibited by o-phenanthroline, 8-hydroxyquinoline, and alpha,alpha'-dipyridyl. Thus, this enzyme was classified into a group of NAD(+)-dependent R-(-)-specific secondary alcohol dehydrogenases. However, this enzyme was better than the previously reported NAD(+)-dependent R-(-)-specific secondary alcohol dehydrogenases for chiral chemical synthesis in terms of substrate specificity, stereospecificity, and thermostability. This enzyme might be applicable as an effective biocatalyst for the production of chiral alcohols and related compounds.