Pseudomonas pickettii DTP0602 utilizes 2,4,6-trichlorophenol (2,4,6-TCP) as a sole source of carbon and energy. 2,4,6-TCP is dechlorinated and converted to 2,6-dichlorohydroquinone by a primarily attacking enzyme of catabolism. The genes encoding the enzyme were cloned using a transposon tagging strategy in Escherichia coil and Pseudomonas putida. A kanamycin-resistant strain derived from P. pickettii DTP0602 by Tn5 insertion, which was named DTP6251, had less dechlorinase activity of 2,4,6-TCP than the parent strain, and 2,6-dihydroquinone accumulated in the culture broth of this mutant. A DNA fragment containing Tn5 together with its flanking region was isolated from strain DTP6251. Deletion and subcloning analysis of the fragment showed that a 3.5-kb region flanked by Tn5 was essential for dechlorinase activity. Two open reading frames denoted hadA and hadB were located in the region : hadA spanned 1,554 nucleotides and encoded a polypeptide with a deduced molecular mass of 58,540; hadB spanned 591 nucleotides and encoded a polypeptide with a deduced molecular mass of 21,167., A set of promoter sequences (sigma(54) recognition sequence; -GG-...-GC- at positions -24 and -12) was found upstream of hadA. Two polypeptides were produced when this region was expressed under the control of the tac and trc promoters in E. coil. HadA was a chlorophenol-4-hydroxylase that hydroxylated various chlorophenols other than 2,4,6-TCP at position 4 to yield corresponding p-dihydroquinones. HadA seemed to be a flavoprotein because FAD and NADH were required for its hydroxylation activity in vitro. Even though both hadA and hadB were essential for expression of hydroxylase activity in vivo, the mixture consisting of HadA and NADH (and/or FAD) expressed hydroxylase activity in vitro. The product of the hadB gene (i.e., HadB) was not essential for expression of dechlorinase activity in vitro.