Reactive oxygen and nitrogen species have been implicated as mediators of mucosal injury in inflammatory bowel disease. This study investigated hydroxyl radical (.OH) generation in the inflamed colon of dextran sulfate sodium (DSS)-induced colitis by measuring the .OH-specific product of salicylate hydroxylation, 2,3-dihydroxybenzoic acid (DHB). Colitis was induced in 6-7 week old CBA/H male mice by supplementing the drinking water with 5% DSS for 7 days. On the last day of dextran exposure, mice were injected with salicylate (SAL) (100 mg/kg i.p.) 60 min before sacrifice, and mucosal homogenates were assayed for SAL and 2,3-DHB by HPLC with fluorescence and electrochemical detection. Mucosal 2,3-DHB levels in mice exposed to 5% DSS were increased by 83% (p < .005); however, SAL levels were also elevated by 182% (p < .001). This translated to a 34% decrease in the ratio 2,3-DHB:SAL in inflamed mucosa, possibly indicating greater catabolism or decreased production of 2,3-DHB. In vitro investigation of the stability of DHBs and SAL in the presence of oxidants of inflammatory lesions revealed that 2,3-DHB and 2,5-DHB were rapidly degraded by hypochlorous acid (HOCl), with initial decomposition rates of 190 and 281 nmol/min, respectively (100microM DHB with 200microM HOCl). Methionine prevented decomposition of DHBs in vitro; however, in mice with 5% DSS-induced colitis, where mucosal myeloperoxidase activity was ten-fold control levels (p < .001), administration of methionine (up to 200 mg/kg i.p.) with SAL was ineffective at increasing the ratio 2,3-DHB:SAL. SAL was also degraded in vitro by HOCl (4.7 nmol/min) resulting in the formation of new fluorescent species which may be useful as indicators of HOCl-mediated injury. Salicylate hydroxylation was unable to provide conclusive evidence supporting a role for .OH in the tissue injury of DSS-induced colitis, as metabolic disturbances in the diseased animals other than changes in .OH generation may have altered 2,3-DHB levels. This problem is relevant to any study involving the in vivo use of trapping molecules. In particular, the susceptibility of 2,3-DHB to degradation by HOCl brings into question the usefulness of salicylate hydroxylation for measurement of .OH-generation in any neutrophilic inflammatory lesion.