Several lines of evidence suggest a role for ACh in the mediation of cerebello-thalamic transmission. The physiological, pharmacological and biochemical experiments described were designed to test this hypothesis for the rat cerebello-thalamic pathway. Unilateral electrolytic lesions of the superior cerebellar peduncle resulted in modest falls of CAT from both ventromedial thalamic nuclei (contralateral 35%, ipsilateral 15%). Iontophoretic application of ACh to relay cells evokes three types of response (i) excitation (ii) inhibition (iii) polyphasic combinations of (i) and (ii). The type of response evoked was directly related to the firing pattern of the cell. Thus, for example, excitatory responses were never recorded during high-frequency bursting but were easily evoked following a switch to tonic, single-spike activity. All responses to ACh and synaptic responses to cerebellar stimulation were sensitive to muscarinic but not to nicotinic cholinergic antagonists. The nicotinic antagonist mecamylamine was a potent blocker of excitant amino acid responses but had no effect on cerebellar-evoked synaptic responses. Cholinergic and anticholinergic agents had a profound action on relay cell firing pattern. ACh promoted single-spike activity whereas atropine promoted high-frequency bursting. The actions of ACh are discussed with reference to recently discovered voltage-sensitive ionic conductances. Because of the modulatory action of ACh on relay cell firing pattern and excitability no firm conclusion can be reached concerning the hypothesis under test here. We tentatively suggest a dual role for ACh as both neurotransmitter and neuromodulator.