Studies have demonstrated different pathogenetic key factors in gentamicin-induced hair cell death. The production of reactive oxygen species (ROS), as well as apoptosis-related genes, play a critical role. However, a coordinated large-scale investigation of gene expression in the organ of Corti (OC) exposed to gentamicin has not yet been conducted. Here we used DNA microarray technology to compare the expression profile of OC exposed to gentamicin to the expression profile of untreated OC. The OCs of Sprague Dawley rats were dissected and the basal turns were cultured. Two-thirds of the explants were then exposed to l00 microM gentamicin, for 4 and 8 h, while one-third of the explants remained in culture medium alone. Gene expression was analyzed using DNA microarray technology and the dChip software package. Based on the results, the 4-h time-point was chosen for further analysis. In these assays, out of 8800 genes, 12 genes were identified on the basis of differential expression in the OC exposed to gentamicin vs. control OC. The identity of these genes suggests that the response of the OC to the gentamicin challenge involves down-regulation of specific gene families in order to alleviate ROS and N-methyl-D-aspartate (NMDA) receptor-mediated cellular stress.