Significant neurochemical, immunocytochemical, and ligand binding studies support a role for GABA as an inhibitory neurotransmitter in the inferior colliculus (IC). The present study attempted to satisfy some of the remaining criteria for establishing transmitter identity by utilizing iontophoretic application onto IC neurons of agents affecting the action of gamma-aminobutyric acid (GABA). The agents examined include GABA, a GABAB agonist (baclofen), a GABAA antagonist (bicuculline), a GABA uptake inhibitor (nipecotic acid), and a benzodiazepine (flurazepam), thought to exert its actions on the GABA receptor complex. Application of GABA results in inhibition of the spontaneous firing and acoustically evoked responses of inferior colliculus neurons. The inhibitory effect of GABA is enhanced by the simultaneous application of nipecotic acid or flurazepam. These agents as well as baclofen produce firing reductions when applied alone in higher doses. The effect of GABA can be blocked by application of bicuculline, and acoustically evoked (binaural) inhibition can also be selectively blocked by low doses of this GABAA antagonist. These data along with previous studies utilizing different techniques fulfill many of the criteria for establishment of GABA as an important inhibitory transmitter in the inferior colliculus.