BACKGROUND AND PURPOSE Exposure to an ototoxic level of an aminoglycoside can result in hearing loss. In this we study investigated the otoprotective efficacy of dexamethasone (DXM), melatonin (MLT) and tacrolimus (TCR) in gentamicin (GM)-treated animals and cultures. EXPERIMENTAL APPROACH Wistar rats were divided into controls (treated with saline); exposed to GM only (GM); and three GM-exposed groups treated with either DXM, MLT or TCR. Auditory function and cochlear surface preparations were studied. In vitro studies of oxidative stress, pro-inflammatory cytokine mRNA levels, the MAPK pathway and caspase-3 activation were performed in organ of Corti explants from 3-day-old rats. KEY RESULTS DXM, MLT and TCR decreased levels of reactive oxygen species in GM-exposed explants. The mRNA levels of TNF-α, IL-1β and TNF-receptor type 1 were significantly reduced in GM + DXM and GM + MLT groups. Phospho-p38 MAPK levels decreased in GM + MLT and GM + TCR groups, while JNK phosphorylation was reduced in GM + DXM and GM + MLT groups. Caspase-3 activation decreased in GM + DXM, GM + MLT and GM + TCR groups. These results were consistent with in vivo results. Local treatment of GM-exposed rat cochleae with either DXM, MLT or TCR preserved auditory function and prevented auditory hair cell loss. CONCLUSIONS AND IMPLICATIONS In organ of Corti explants, GM increased oxidative stress and initiated an inflammatory response that led to the activation of MAPKs and apoptosis of hair cells. The three compounds tested demonstrated otoprotective properties that could be beneficial in the treatment of ototoxicity-induced hearing loss.