The potential role of nitric oxide (NO) in iron-induced toxicity was studied in proximal tubule cells in primary culture. NO production (NO2-/NO3-) was significantly increased in iron-treated compared with control cells (3.43 +/- 0.08 vs. 1.56 +/- 0.08 nmol/dish, P < 0.01). NO synthase (NOS) activity was also induced by iron treatment (16.2 +/- 2.0 vs. 0.4 +/- 0.2 nmol of [3H]citrulline/mg protein, P < 0.01). L-Arginine, a substrate for NOS, augmented iron-induced NO production and cell damage [lactate dehydrogenase (LDH) leakage], whereas aminoguanidine, an inhibitor of NOS, reduced iron-induced NO production and LDH leakage. Sodium nitroprusside, an exogenous NO donor, induced LDH leakage in a dose-dependent manner, but no effect on lipid peroxidation (malo-ndialdehyde bis[dimethyl acetal] (MDA) production) was observed. Superoxide dismutase and catalase decreased iron-induced MDA production but did not affect LDH leakage or NO production. Dimethylpyrroline N-oxide and desferal prevented MDA production, LDH leakage, and NO production. We concluded that NO is one of the mediators of iron-induced toxicity in proximal tubule cells. NO-induced toxicity is not dependent on lipid peroxidation. This may explain the variable effect of different antioxidants on cell damage and lipid peroxidation in iron-induced cytotoxicity.