Chlorobenzene cis-dihydrodiol dehydrogenase was purified to homogeneity from Xanthobacter flavus 14p1, which used 1,4-dichlorobenzene as the sole source of carbon and energy. The enzyme converted a number of halogenated substrates with high specific activity. The pI of the native chlorobenzene cis-dihydrodiol dehydrogenase was 5.4, and the molecular mass was approximately 100 kDa, as determined by gel filtration. The enzyme was composed of four apparently identical subunits with a molecular mass of 26.5 kDa. The Michaelis constant for 3,6-dichlorobenzene cis-dihydrodiol (210 microM) was lower than for benzene cis-dihydrodiol (780 microM), while the specific activity with benzene cis-dihydrodiol (63 units/ mg) was higher than with 3,6-dichlorobenzene cis-dihydrodiol (32 units/mg). Chlorobenzene cis-dihydrodiol dehydrogenase accepted also NADP+ as cosubstrate; however, the activity was reduced to 14% of that with NAD+. The enzymic activity was inhibited by mercuric chloride and to a lesser extent by the metal-ion chelators 8-hydroxyquinoline and KCN.