The proposal that free-radical generation contributes to the ototoxicities of several chemical agents was studied utilizing electron paramagnetic resonance (EPR) spectrometry to detect directly ototoxicant-induced reactive oxygen species formation in cochlear tissue. Guinea pig cochlear explants in chelexed artificial perilymph (AP: 200 microliters) were exposed to an ototoxicant or AP for 10 min. Ototoxic agents included gentamicin sulfate (4.0 mM), kanamycin monosulfate (4.0 mM), ethacrynic acid (0.5 mM), furosemide (0.3 mM), cisplatin (0.1 mM), trimethyltin chloride (0.1 mM), and quinine HCl (3.0 mM). Following incubation, 20 microliters of AP/ototoxicant mixture was replaced by the filtered spin trap, 5,5-dimethylpyrroline-N-oxide (DMPO). After 10 min, the EPR spectrum of the mixture was obtained. Four line EPR spectra of relative intensities 1:2:2:1, associated with hydroxyl radical (OH)/DMPO adduct formation, were evidenced by reaction mixtures containing cochlear explants exposed to each ototoxicant. Cisplatin, quinine and the loop diuretics produced weak OH-associated EPR signals in the absence of a cochlear explant, which were amplified in its presence. Deferoxamine quenched all OH spectral peaks. Peroxide levels, assayed in parallel experiments, were diminished by each ototoxicant relative to those seen following AP exposure, suggesting possible H2O2 conversion to OH. These data support the proposal that various ototoxic agents are capable of reactive oxygen species generation or promotion in cochlear tissues.