In vitro experiments were performed in the superfused brainstem-spinal cord preparation of newborn rats in order to analyse the central respiratory effects of acetylcholine. The central motor output was assessed from recording electrical activity in nerves supplying respiratory muscles. Acetylcholine added to the bathing medium induced dose-dependent increases in respiratory frequency which were blocked by muscarinic (but not nicotinic) antagonists and enhanced by physostigmine. These effects originated from the medullary ventral surface where chemosensitive structures have been previously located. The respiratory central chemosensitivity of the isolated brainstem was analysed using a CO2 free, pH 7.9 medium instead of the normal medium (bubbled with 5% CO2, pH 7.3). Decreases at the H+ and CO2 stimuli led to decreased inspiratory activity, resulting mainly from a decrease in the amplitude of the motor output. These responses were enhanced by atropine and diminished by physostigmine. These results obtained in vitro on the newborn rat suggest that cholinergic synapses are not directly involved in the genesis of respiratory rhythmicity but confirm previous results obtained in vivo in adult animal revealing that acetylcholine is implicated in the central respiratory chemosensitivity.