Iron overload with ferric nitrilotriacetate (Fe-NTA) induces acute renal proximal tubular necrosis, a consequence of oxidative tissue damage, that eventually leads to a high incidence of renal adenocarcinoma in rodents. A recent immunohistochemical study has revealed that the materials immunoreactive to the antibody against the aldehyde (4-hydroxy-2-nonenal)-modified proteins are generated in the renal proximal tubules of rats treated with Fe-NTA (S. Toyokuni et al. (1994) Proc. Natl. Acad. Sci. USA 91, 2616-2620). Here we present further evidences that cytosolic proteins modified with lipid peroxidation-derived aldehydes (4-hydroxy-2-nonenal and malondialdehyde) are indeed accumulated in the kidney of rats treated with Fe-NTA. A single intraperitoneal Fe-NTA treatment (15 or 30 mg Fe/kg body wt) induced a rapid accumulation of thiobarbituric acid-reactive substances, a direct consequence of membrane lipid peroxidation, mostly within the cytosolic compartment of rat kidney. The accumulation of lipid peroxidation-derived aldehydes was associated with an apparent accumulation (from 67.9 nmol in untreated controls to 83.3 nmol/mg protein in Fe-NTA-treated rats at 3 h after treatment) and subsequent elimination of aberrant proteins as assessed by a 2,4-dinitrophenylhydrazine postlabeling assay. Immunoblot analysis with two different polyclonal antibodies against 4-hydroxy-2-nonenal-modified and malondialdehyde-modified proteins detected a temporary accumulation of aldehyde-modified proteins in the cytosol. An immunohistochemical technique with serial sectioning revealed that immunoreactivities of these antibodies were observed in the identical portion of the renal proximal tubules. The implications of these data in relation to the nephrotoxicity of Fe-NTA are discussed.