In the inner ear, the excitatory amino acid glutamate is a proposed neurotransmitter acting at the synapse between hair cells and afferent auditory neurons. Using cultures of 5-day-old rat auditory neurons, we show that the afferent auditory neuronal population can be divided, on the basis of its sensitivity to the neuronotoxic effect of glutamate and its analogs, in at least 3 subpopulations, one responding to N-methyl-D-aspartate (NMDA), one responding to kainate and a third minor one unresponsive to NMDA, kainic acid and glutamate. No toxic effect of quisqualate is observed. The use of specific antagonists (kynurenate and 2-amino-5-phosphonovalerate (DAP-5) demonstrates the specificity of the receptors to the excitatory amino acids on the afferent auditory neurons. Afferent auditory neurons from adult rats can also be cultured and in these preparations only the large neurons are sensitive to glutamate, kainate and NMDA while the small neurons are not responsive, suggesting that a glutamatergic neurotransmission occurs only at this synapse between the inner hair cells and the large radial afferent auditory neurons. We also show that, in vitro, the organ of Corti releases, in response to an increased potassium concentration and in the presence of calcium, a toxic activity for the afferent auditory neurons that is antagonized by kynurenate and DAP-5. Pathophysiological implications are discussed.