Reactive oxygen species (ROS) have been suggested to play a major role in aminoglycoside-induced hair cell (HC) loss, but are difficult to detect. Moreover, ROS can occur normally in cells where they have roles in metabolism, cell signaling and other processes. Two new probes, aminophenyl fluorescein (APF) and hydroxyphenyl fluorescein (HPF) are dyes which selectively detect highly-reactive oxygen species (hROS), those most associated with cellular damage. We assessed the presence of hROS in the neonatal rat organ of Corti during chronic exposure to 50 microM gentamicin in vitro, to examine the relationship between cell damage and hROS across HC type and across the three cochlear turns. hROS were initially detected at 48 hours (h), with an increase at 72 h and persistence until at least 96 h. At 48 h, hROS were restricted to outer HCs and occurred prior to loss of stereocilia. At 72 h, outer HCs showed both hROS and stereocilia loss, and hROS were noted in a few inner HCs. Basal turn HCs showed more hROS than middle turn HCs. Very little hROS accumulation or stereocilia loss was observed in the apical turn, even at 72 h. First row outer HCs were most vulnerable to gentamicin-induced hROS, followed by second and then third row outer HCs. Inner HCs behaved similarly to third row outer HCs. By 96 h stereocilia damage was extensive, but surviving HCs showed persisting fluorescence. APF consistently showed more fluorescence than HPF. The results suggest that hROS accumulation is an important initial step in gentamicin-induced HC damage, and that the differential sensitivity of HCs in the organ of Corti is closely related to differences in hROS accumulation.