Participation of free radicals in the adverse renal and cochlear side effects of aminoglycoside antibiotics is controversial. We measured the production of free radicals by gentamicin in vitro through the oxidation of arachidonic acid. Gentamicin alone (0.05 to 10 mM) did not cause lipid peroxidation. However, it dramatically promoted radical formation in the presence of iron salts. Peroxidation was maximal at 1 mM gentamicin plus 0.1 mM Fe(II)/Fe(III) (0.05 mM FeSO₄ and FeCl₃ each). At these iron concentrations, peroxidation was not significant in the absence of gentamicin. Since chelators can enhance iron-catalyzed oxidations, this finding suggested that gentamicin-dependent radical formation was based upon iron chelation. This hypothesis was tested by measuring the influence of gentamicin on the oxidation of salicylate by Fe-EDTA complexes, a reaction that is inhibited by competing iron chelators. Gentamicin was a concentration-dependent inhibitor. In contrast, concentrations of gentamicin as high as 50 mM did not interfere with iron-independent salicylate oxidation. These results suggest that gentamicin acts as an iron chelator, and that the iron-gentamicin complex is a potent catalyst of free radical formation.