The involvement of cytosolic nitric oxide (NO) and mitochondrial superoxide radical (O2(.-)) production was evaluated as a mechanism triggering liver oxidative stress in lindane (40 mg/kg) or L-3,3',5-triiodothyronine (T3, 0.1 mg/kg for 2 consecutive days) treated animals (male Sprague-Dawley rats) subjected to iron overload (200 mg/kg). Lindane and iron led to 504 and 210% increases in the content of hepatic protein carbonyls as an index of oxidative stress, with a 706% enhancement being produced by their combined administration. T3 did not alter this parameter, whereas iron overload increased the content of protein carbonyls by 116% in hyperthyroid rats. Lindane increased NO generation by 106% without changes in generation of O2(.-), whereas iron enhanced both parameters by 109 and 80% over control values, respectively, with a net 33 and 46% decrease, respectively, being elicited by the combined treatment related to iron overload alone. Hyperthyroidism increased liver NO (69%) and O2(.-) (110%) generation compared to controls, effects that were either synergistically augmented or suppressed by iron overload, respectively. The in vitro addition of iron (1 micromol/mg protein) to liver cytosolic fractions from euthyroid (97%) and hyperthyroid (173%) rats also enhanced NO generation. The effects of iron overload on mitochondrial O2(.-) production by hyperthyroid rats were reproduced by the in vitro addition of 1 micromol iron/mg protein and abolished by the in vivo pretreatment with the iron chelator desferrioxamine (500 mg/kg). It is concluded that liver oxidative stress induced by iron overload is independent of NO and O2(.-) production in lindane-treated rats, whereas in hyperthyroid animals NO generation is a major factor contributing to this redox imbalance.