Kinetic and structural studies have shown that peroxidases are capable of the oxidation of 2,4,6-trichlorophenol (2,4,6-TCP) to 2,6-dichloro-1,4-benzoquinone (2,6-DCQ). Further reactions of 2,6-DCQ in the presence of H2O2 and OH- yield 2,6-dichloro-3-hydroxy-1,4-benzoquinone (2,6-DCQOH). The reactions of 2,6-DCQ have been monitored spectroscopically [UV visible and electron spin resonance (ESR)] and chromatographically. The hydroxylation product, 2,6-DCQOH, has been observed by UV visible and characterized structurally by H-1 and C-13 NMR spectroscopy. The results are consistent with a nonphotochemical base-catalyzed oxidation of 2,6-DCQat pH >7. Because H2O2 is present in peroxidase reaction mixtures, there is also a potential role for the hydrogen peroxide anion (HOO-). However, in agreement with previous work, we observe that the nonphotochemical epoxidation by H2O2 at pH < 7 is immeasurably slow. Both room-temperature ESR and rapid-freeze-quench ESR methods were used to establish that the dominant nonphotochemical mechanism involves formation of a semiquinone radical (base -catalyzed pathway), rather than epoxidation (direct attack by H2O2 at low pH). Analysis of the kinetics using an Arrhenius model permits determination of the activation energy of hydroxylation (E-a = 36 kJ/mol), which is significantly lower than the activation energy of the peroxidase-catalyzed oxidation of 2,4,6-TCP (E-a = 56 kJ/mol). However, the reaction is second order in both 2,6-DCQ and OH- so that its rate becomes significant above 25 degrees C due to the increased rate of formation of 2,6-DCQ that feeds the second-order process. The peroxidase used in this study is the dehaloperoxidase-hemoglobin (DHP A) from Amphitrite ornata, which is used to study the effect of a catalyst on the reactions. The control experiments and precedents in studies of other peroxidases lead to the conclusion that hydroxylation will be observed following any process that leads to the formation of the 2,6-DCQ at pH > 7, regardless of the catalyst used in the 2,4,6-TCP oxidation reaction.