SUMMARY1. Presynaptic inhibition was evoked in the rat cuneate nucleus by a peripheral conditioning stimulus. The dicarboxylic amino acid salts glutamate and aspartate and the neutral amino acids glycine and yaminobutyric acid (GABA) were topically applied to a restricted area of the cuneate nucleus and their effects on both resting primary afferent terminal excitability and the increase in excitability of afferent terminals during presynaptic inhibition determined.2. Aspartate had no effect on either resting primary afferent terminal excitability or on the increase in excitability during presynaptic inhibition.3. Glycine reduced both resting primary afferent terminal excitability and presynaptic inhibition.4. Glutamate increased both resting primary afferent terminal excitability and presynaptic inhibition while GABA increased resting primary afferent terminal excitability but reduced the increase in excitability during presynaptic inhibition.5. The convulsant alkaloids picrotoxin (given intravenously) and bicuculline (topically applied) blocked presynaptic inhibition. The blocking action of picrotoxin was overcome by topical application of GABA but not glutamate.6. Simultaneous measurement of pre-and post-synaptic excitability in the cuneate nucleus showed that while glutamate increased excitability at both sites, GABA increased primary afferent terminal excitability but depressed post-synaptic excitability.7. It is concluded that glycine and glutamate exert non-specific actions on primary afferent terminals similar to their effects at post-synaptic sites elsewhere in the c.N.s. while GABA depolarizes primary afferent terminals by a specific action at the same receptor site as the presynaptic inhibitory transmitter. The possibility is discussed that the presynaptic inhibitory transmitter in the cuneate nucleus is GABA or a closely related substance.