Chronic iron overload (CIO) enhances nitric oxide (NO) production in the liver, which may represent a hepatoprotective mechanism against CIO toxicity. In order to test this hypothesis, the influence of CIO (diet enriched with 3% (wt/wt) carbonyl-iron for 8 weeks) in the absence or presence of the NO synthase (NOS) inhibitor N G -nitro-l-arginine methyl ester (l-NAME) on NOS activity, extracellular signal-regulated kinase (ERK1/2) and NF-κB activation was studied, in relation to ferritin expression and liver morphology. CIO increased liver NOS activity, ERK1/2 phosphorylation, NF-κB DNA binding, and ferritin expression, with normal liver histology. These changes were suppressed by combined CIO and l-NAME treatment, with the resulting inflammatory response of the liver. It is concluded that NO response induced by CIO represents a molecular mechanism affording protection against iron toxicity, which is related to both the activation of the ERK/NF-κB pathway involving inducible NOS expression and ferritin upregulation, changes that may be interrelated.