Objective and methods: Eupnoic breathing in mammals is generated and modulated in a respiratory network localised in the ponto-medullary brainstem. Different respiratory neurons are coupled via various synaptic connections and use a multiplicity of neurotransmitters to coordinate the activity of cranial and spinal motor outputs. The role of acetylcholine (ACh) in the central control of breathing is not yet elucidated. Different subtypes of acetylcholine receptors are expressed in respiratory related areas in the brainstem. This might explain the effects of organophosphorus compounds (OPs) that lead to accumulation of ACh in the periphery and central in the brain. We investigated the effects of specific nicotinic and muscarinic ACh antagonists and agonists on the central breathing pattern in the in situ arterially perfused brainstem preparation of rat. This preparation generates an eupnoeic like breathing pattern, which can be recorded on phrenic nerve activity. Results: No effect was observed for the tested antagonists under normal conditions, without applications of additional chemicals. One exception was TMPH-hydrochloride the only tested non- competitive antagonist, which blocks the neuronal nicotinic AChRs. Application caused an elongation of the cycle length, reduction of the frequency and a decline in tidal volume. Competitive antagonists like telenzepine-dihydrochloride which selectively blocks M1 muscarinic AChRs, exert influence only after application of carbachol, a non-selective ACh agonist. Conclusions: A block of AChRs under physiological ACh concentrations does not influence breathing except a non- competitive antagonist is applied. If the level of ACh is elevated via carbachol, block of AChR results in a fast recovery and re- establishes eupnoic breathing. It awaits further analysis if the effects are the same when the ACh level is increased by intoxication with OPs. (Supported by contract research project for the Bundeswehr Medical Service)