Iron imbalance has been implicated in oxidative injury associated with many brain diseases. The present study investigated the importance of iron location in hydroxyl radical (.OH) generation and the link between .OH production evaluated by the salicylate method and lipid peroxidation monitored by thiobarbituric acid-reactive substances assay. Brain slices were exposed to increasing doses (2, 10 and 50 microM) of Fe(III) that was complexed either to a lipophilic (8-hydroxyquinoline, HQ) or to a hydrophilic (ammoniacal citrate) ligand. Both iron complexes resulted in an increased salicylate hydroxylation and lipid peroxidation, these effects being significantly more potent in presence of Fe(III)-HQ. Salicylate hydroxylation was linearly correlated to the intensity of TBARS formation but the slope of the curve was found to be higher with Fe(III)-HQ. The present results demonstrate that 1) cell-associated reactive iron is more prone than extracellular iron to induce .OH generation, 2) the level of lipid peroxidation depending on the site of .OH production, cannot be used as an index of the level of total .OH formation, 3) the salicylate method is a convenient method to detect .OH formed intracellularly, at least in vitro.