In vivo bilateral microdialysis in the rat striatum was used to investigate hydroxyl radical formation under basal conditions and after intrastriatal administration of the neurotoxin, 1-methyl-4-phenylpyridinium (MPP+). After a short equilibration period, 4-hydroxybenzoate (4HBZ), which scavenges hydroxyl radicals to produce 3,4-dihydroxybenzoate (34DHB), was injected intraperitoneally 15 min before infusion of MPP+. To evaluate the enzymatic contribution to hydroxyl radical formation, two other series of microdialyses were performed following administration of monoamine oxidase B inhibitors, either 1-deprenyl (selegiline) or MDL 72,974A [(E)-2-(4-fluorophenethyl)-3-fluoroallylamine hydrochloride]. Microdialysate samples were analyzed by high-performance liquid chromatography for catecholamines, 3,4-dihydroxyphenylacetate (DOPAC), homovanillate (HVA), along with the hydroxyl radical adduct, 34DHB and its precursor, 4HBZ. MPP+ administration resulted in a massive release of dopamine along with a decrease in DOPAC and HVA in all three groups. A striking effect in all three groups was noted in which MPP+ resulted in a decrease in interstitial 4HBZ to < 50% of the non-MPP+ -treated side. In absolute terms, the amount of 34DHB produced was low but similar in all three groups, even after unilateral MPP+ infusion. When 34DHB was normalized to 4HBZ release to account for differences in precursor availability, there were no significant differences in the 34DHB/4HBZ ratios either with or without MAO inhibitor treatment or after local MPP+ infusion. Systemic 4HBZ administration appears to result predominantly in intra-cellular sampling of hydroxyl radicals which produces different results from local infusion of trapping agents such as salicylate.