Haloalkane dehalogenases are enzymes catalysing the cleavage of the carbon-halogen bond by a hydrolytic mechanism. Genomes of Mycobacterium tuberculosis and M. bovis contain at least two open reading frames coding for the polypeptides showing a high sequence similarity with biochemically characterised haloalkane dehalogenases. In this paper we have described the cloning of the haloalkane dehalogenase genes dmbA and dmbB from M. bovis 5033/66 and proved the dehalogenase activity of their translation products. Both these genes are widely distributed among species of the M. tuberculosis complex, including M. bovis, M. bovis BCG, M. africanum, M. caprae, M. microti, and M. pinnipedii, as shown by the PCR screening of forty eight isolates from various hosts. DmbA and DmbB proteins were heterologously expressed in Escherichia coli and purified to homogeneity. The DmbB protein had to be expressed in a fusion with thioredoxin to obtain a soluble protein sample. The temperature optimum of DmbA and DmbB proteins determined with 1,2-dibromoethane is 45C. The melting temperature assessed by circular dichroism spectroscopy of DmbA is 47C and DmbB is 57C. The pH optimum of DmbA depends on composition of a buffer with maximal activity at 9.0. DmbB had a single pH optimum at pH 6.5. Mycobacteria are currently the only genus known to carry more than one haloalkane dehalogenase gene although putative haloalkane dehalogenases can be inferred in more then 20 different bacterial species by comparative genomics. Evolution and distribution of haloalkane dehalogenases among mycobacteria is discussed.