Ralstonia eutropha JMP134 can grow on several chlorinated aromatic pollutants, including 2,4-dichlorophenoxyacetate and 2,4,6-trichlorophenol (2,4,6-TCP). Although a 2,4,6-TCP degradation pathway in JMP134 has been proposed, the enzymes and genes responsible for 2,4,6-TCP degradation have not been characterized. In this study, we found that 2,4,6-TCP degradation by JMP134 was inducible by 2,4,6-TCP and subject to catabolic repression by glutamate. We detected 2,4,6-TCP-degrading activities in JMP134 cell extracts. Our partial purification and initial characterization of the enzyme indicated that a reduced flavin adenine dinucleotide (FADH2)-utilizing monooxygenase converted 2,4,6-TCP to 6-chlorohydroxyquinol (6-CHQ). The finding directed us to PCR amplify a 3.2-kb fragment containing a gene cluster (tcpABC) from JMP134 by using primers designed from conserved regions of FADH2-utilizing monooxygenases and hydroxyquinol 1,2-dioxygenases. Sequence analysis indicated that tcpA, tcpB, and tcpC encoded an FADH2-utilizing monooxygenase, a probable flavin reductase, and a 6-CHQ 1,2-dioxygenase, respectively. The three genes were individually inactivated in JMP134. The tcpA mutant failed to degrade 2,4,6-TCP, while both tcpB and tcpC mutants degraded 2,4,6-TCP to an oxidized product of 6-CHQ. Insertional inactivation of tcpB may have led to a polar effect on downstream tcpC, and this probably resulted in the accumulation of the oxidized form of 6-CHQ. For further characterization, TcpA was produced, purified, and shown to transform 2,4,6-TCP to 6-CHQ when FADH2 was supplied by an Escherichia coli flavin reductase. TcpC produced in E. coli oxidized 6-CHQ to 2-chloromaleylacetate. Thus, our data suggest that JMP134 transforms 2,4,6-TCP to 2-chloromaleylacetate by TcpA and TcpC. Sequence analysis suggests that tcpB may function as an FAD reductase, but experimental data did not support this hypothesis. The function of TcpB remains unknown.