Burkholderia cepacia AC1100 metabolizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) via formation of 5-chlorohydroxyquinol (5-CHQ), hydroxyquinol (HQ), maleylacetate, and beta-oxoadipate. The step(s) leading to the dechlorination of 5-CHQ to HQ has remained unidentified. We demonstrate that a dechlorinating enzyme, TftG, catalyzes the conversion of 5-CHQ to hydroxybenzoquinone, which is then reduced to HQ by a hydroxybenzoquinone reductase (HBQ reductase). HQ is subsequently converted to maleylacetate by hydroxyquinol 1,2-dioxygenase (HQDO). All three enzymes were purified. We demonstrate specific product formation by colorimetric assay and mass spectrometry when 5-CHQ is treated successively with the three enzymes: TftG, TftG plus HBQ reductase, and TftG plus HBQ reductase plus HQDO. This study delineates the complete enzymatic pathway for the degradation of 5-CHQ to maleylacetate.