The functional role of GABA and glycine in monaural and binaural signal analysis was studied in single unit recordings from the central nucleus of the inferior colliculus (IC) of horseshoe bats (Rhinolophus rouxi) employing microiontophoresis of the putative neurotransmitters and their antagonists bicuculline and strychnine. Most neurons were inhibited by GABA (98%; N = 107) and glycine (92%; N = 118). Both neurotransmitters appear involved in several functional contexts, but to different degrees. Bicuculline-induced increases of discharge activity (99% of cells; N = 191) were accompanied by changes of temporal response patterns in 35% of neurons distributed throughout the IC. Strychnine enhanced activity in only 53% of neurons (N = 147); cells exhibiting response pattern changes were rare (9%) and confined to greater recording depths. In individual cells, the effects of both antagonists could markedly differ, suggesting a differential supply by GABAergic and glycinergic networks. Bicuculline changed the shape of the excitatory tuning curve by antagonizing lateral inhibition at neighboring frequencies and/or inhibition at high stimulation levels. Such effects were rarely observed with strychnine. Binaural response properties of single units were influenced either by antagonization of inhibition mediated by ipsilateral stimulation (bicuculline) or by changing the strength of the main excitatory input (bicuculline and strychnine).