The cholinergic modulation of the electrical properties of spinal motoneurones was investigated in vitro, with the use of the whole-cell patch-clamp recording technique in lumbar spinal cord slices from juvenile urodeles (Pleurodeles waltlii). Bath application of acetylcholine (20 μm) with eserine (20 μm) induced an increase in the resting membrane potential, a decrease of the input resistance, a decrease of the action potential amplitude, and a reduction of the medium afterhyperpolarization (mAHP) that followed each action potential. Moreover, the firing rate of motoneurones during a depolarizing current pulse and the slope of their stimulus current–spike frequency relation were increased. All of these effects were mimicked by extracellular application of muscarine (20 μm), and blocked by application of the muscarinic receptor antagonist atropine (0.1–1 μm). They were not observed during bath application of nicotine (10 μm). These results suggest that the cholinergic modulation of spinal motoneurone excitability was mediated by activation of muscarinic receptors. Our results further show that the muscarinic action primarily resulted from a reduction of the Ca2+-activated K+ current responsible for the mAHP, an inhibition of the hyperpolarization-activated cation current, Ih, and an enhancement of the inward rectifying K+ current, IKir. We conclude that cholinergic modulation can contribute significantly to the production of motor behaviour by altering several ionic conductances responsible for the repetitive discharge of motoneurones.