The extracellular level of neurotransmitter amino acids and total content of serotonin and its metabolite 5-HIAA were estimated by microdialysis in cerebellar vermis at different stages of habituation of the acoustic startle reaction (ASR) and conditioned freezing in a startle chamber prior to acoustic stimulation. An increase in GABA and serotonin levels was observed 10 min after training. Twenty four hours after training, glycine and glutamate levels increased, whereas serotonin and 5-HIAA contents decreased. Positive correlations were revealed between the GABA and glycine levels during training and characteristics of the freezing behavior during testing. Glutamate level during the test session positively correlated with the index of long-term habituation of the ASR orienting component. NMDA receptor antagonist APV applied on cerebellar vermis before training suppressed, whereas AM PA receptor antagonist CNQX enhanced the long-term habituation of the ASR orienting component. Under the same conditions, GABA receptor antagonists bicuculline and phaclofen suppressed the long-term habituation of the defensive ASR component and formation of the conditioned freezing behavior. Applied before training 5-HT antagonist ritanserin impaired long-term habituation of defensive ASR component and formation of conditioned freezing behavior, but its application 1 h before testing enhanced the habituation of the defensive ASR component without any effect on conditioned fear. The results testify to the involvement of glutamatergic cerebellar system activity predominantly in long-term habituation of orienting ASR component and participation of GABA interneurons and serotoninergic projections to cerebellum in the mechanisms of the long-term habituation of the defensive ASR component and formation of conditioned freezing behavior.