The present studies were aimed at investigating the possible roles of dopamine (DA) and iron in production of hydroxyl radicals (OH) in rat striatum after Mn2+ intoxication. For this purpose, DA depletions were assessed concomitant with in vivo 2,3- and 2,5-dihydroxybenzoic acid (DHBA) formation from the reaction of salicylate with OH, of which 2,3-DHBA is a nonenzymatic adduct. Following intrastriatal Mn2+ injection, marked 2,3-DHBA increases were observed in a time- and dose-dependent fashion reaching maximum levels at 6-18 h and a plateau beyond 0.4 micromol (fourfold increase). The delayed increase of 2,3-DHBA levels suggestS that Mn2+ induces OH formation in the living brain by an indirect process. The early DA depletion (2 h) and relatively late OH formation (6 h) indicate independent processes by Mn2+. In addition, depletion of DA (about 90%) by reserpine pretreatment not significantly alter Mn2+-induced 2,3-DHBA formation or the extent of DA depletion, suggesting that DA or DA autoxidation are not participating in Mn2+-induced OH formation in vivo. Furthermore, Mn2+ injection did not significantly alter the low molecular weight weight iron pool in striatum, and co-injections of the iron-chelator deferoxamine with Mn(2+) into striatum did not significantly attenuate Mn(2+)-induced 2,3-DHBA formation. These findings suggest no role of chelatable iron in generation of Mn(2+)-induced OH, but do not exclude a role for mitochondrial heme-iron or peroxynitrite (Fe-indepeNdent) in Mn2+-induced OH formation.