Reactive oxygen species (ROS) generation may play a role in ototoxicity, however, the specific effects of ROS generation upon cochlear function are unstudied. Therefore, guinea pig cochleas were instilled with artificial perilymph (AP), H2O2, or confirmed generating systems for the superoxide anion (O2-) or the hydroxyl radical (OH.), or with an ROS system plus its respective scavenger -catalase (CAT), superoxide dismutase (SOD) or deferoxamine (DEF). O2- generating system instillation led to significantly greater mean high frequency compound action potential (CAP) threshold shifts at 10 and 120 min post infusion than seen in AP control or SOD/O2- groups. H2O2 group CAP threshold shifts were significantly greater than control and CAT/H2O2 group values at 10 (16-30 kHz), and 120 min (above 12 kHz). OH generating system instillation led to significantly greater CAP threshold shifts at 10 (12-30 kHz) and 120 min (above 6 kHz) than seen in control or DEF/OH groups. No significant CAP differences were found between controls and scavenger/ROS groups. Mean 1.0 microV cochlear microphonic isopotential curve shift values did not systematically differ among groups. The rapid degradation of high frequency CAP threshold sensitivity seen here may provide insight into the portion of cochlear dysfunction which is ROS-mediated following noise, radiation or chemical exposures.