The use of aminoglycoside antibiotics is limited by ototoxicity that can produce permanent hearing loss. We report that concurrent administration of N–methyl–D–aspartate (NMDA) antagonists markedly attenuates both the hearing loss and destruction of cochlear hair cells in guinea pigs treated with aminoglycoside antibiotics. These findings indicate that aminoglycoside–induced hearing loss is mediated, in part, through an excitotoxic process. The high correlation (Spearman correlation coefficient: 0.928; P < 0.01) obtained between the relative cochleotoxicities of a series of aminoglycosides in humans and the potencies of these compounds to produce a polyamine–like enhancement of [³H]dizocilpine binding to NMDA receptors is consistent with this hypothesis, and provides a simple in vitro assay that can predict this aspect of aminoglycoside–induced ototoxicity.