The effects of acetylcholine (ACh) in processing acoustical information in the inferior colliculus (IC) of awake horseshoe bats (Rhinolophus rouxi) were examined with single cell recordings and microiontophoresis. Cholinergic agonists, acetylcholine and carbachol raised the stimulus evoked discharge in 37% and suppressed responses in 16% of the sample. They did not alter the shapes of tuning curves and rate-intensity functions but the latter showed parallel shifting. The nicotinic antagonist, hexamethonium raised neuronal activity in 52% of neurons without affecting discharge patterns. The nonspecific muscarinic antagonist atropine was mostly inhibitory (62% of units) and caused changes in temporal discharge patterns by affecting the tonic response component. The selective muscarinic ml antagonist pirenzepine, also had an inhibitory effect (37% of units) and predominantly influenced the tonic response component. The selective m2 antagonist, gallamine however produced mainly excitatory effects (64% of units) and changed temporal discharge patterns by adding tonic response components. These findings may indicate a differential pre- and postsynaptic synaptic distribution of m1/m2 receptors in the inferior colliculus as reported for other brain structures. The results indicate that ACh plays a neuromodulatory transmitter role in the auditory midbrain by setting the level of neuronal activity. Its exact function in particular behavioral contexts remains to be determined, since the origin of cholinergic innervation of the mammalian IC is still unclear.