The production of hydroxyl radicals by tetrachlorohydroquinone (TCHQ, a major metabolite of the widely used biocide pentachlorophenol) in the presence of H2O2 was studied by salicylate hydroxylation method. HPLC with electrochemical detection was used to measure the levels of 2,3-and 2,5-dihydroxybenzoic acid (DHBA) formed when the hydroxyl radicals react with salicylate. We found that TCHQ and H2O2 could produce both 2,3- and 2,5-DHBA when incubated with salicylate. Their production was markedly inhibited by hydroxyl radical scavenging agents dimethyl sulfoxide and ethanol, as well as by tetrachlorosemiquinone radical scavengers desferrioxamine and other hydroxamic acids. In contrast, their production was not affected by the nonhydroxamate iron chelators diethylenetriaminepentaacetic acid (DTPA), bathophenanthroline disulfonic acid, and phytic acid, as well as the copper-specific chelator bathocuprione disulfonic acid. A comparison of product formation and distribution from the reaction of ferrous iron with hydrogen peroxide (the classic Fenton system) strongly suggests that the same hydroxyl radical adducts are formed as in the TCHQ/H2O2 experiments. Taken together, we propose that hydroxyl radicals were produced by TCHQ in the presence of H2O2, probably through a metal-independent organic Fenton reaction.