In order to investigate the possible role of glutamate (Glu) as afferent transmitter in the vestibular system, this agent was tested on sensory organs of frog semicircular canals. Intracellular recordings from single afferent axons in isolated labyrinths showed that, after blocking chemical transmission with high Mg++ (12 mM), micro-injections of Glu (5 mM-15 ul) elicited a long lasting postsynaptic depolarization. The amplitude of this depolarization was reduced dose dependently after addition of the amino acid antagonists Kynurenic acid or gamma-D-glutamylglycine to the bath. When the Na+ concentration in the bath was progressively reduced, the depolarization decreased gradually and disappeared almost completely in Na(+)-free Ringer. The removal of K+ affected the depolarization to a lesser extent: in K(+)-free Ringer depolarization decreased only by 30-40%. On the contrary, the complete substitution of Ca++ ions in the bath was without effect. Our results suggest that in the frog semicircular canals the postsynaptic depolarization induced by Glu involves the activation of non NMDA type of amino acid receptors, probably coupled to channels selective for Na+ and K+ ions. The present findings are consistent with the hypothesis that Glu or a related substance may be the transmitter released at the afferent synapses of the vestibular receptors.