Giant neurones in the caudal pontine reticular nucleus (PnC) play a crucial role in mediating the mammalian startle response. They receive input from cochlear, trigeminal and vestibular nuclei and project directly to motoneurones. Furthermore, they integrate modulatory input from different brain regions either enhancing or inhibiting startle responses. One prominent startle modulation is prepulse inhibition where a non-startling stimulus presented prior to the startle stimulus inhibits a subsequent startle response. Several behavioural studies have indicated that this inhibition is mediated by muscarinic receptors at the level of the PnC. Here, we performed whole-cell patch-clamp recordings from PnC giant neurones in acute rat brain slices in order to examine muscarinic inhibition. We stimulated afferent trigeminal and auditory fibres and applied muscarinic agonists and antagonists in order to investigate their effect on excitatory postsynaptic current amplitudes, paired-pulse ratio and passive membrane properties of PnC giant neurones. The cholinergic agonist carbachol and the muscarinic agonist oxotremorine significantly reduced excitatory postsynaptic current amplitudes and increased the paired-pulse ratio. Carbachol additionally reduced the membrane resistance of postsynaptic PnC giant neurones. The subtype-specific antagonists AF-DX116 (M2 preferring) and tropicamide (M4 preferring) antagonized the oxotremorine effect indicating that M4 and possibly M2 receptor subtypes are involved in this inhibition. The G-protein-activated inward rectifying potassium channel blocker tertiapin-Q had no effect on oxotremorine-induced inhibition of giant neurones. Our results show a mainly presynaptically mediated strong inhibition of PnC giant neurones by activation of M4 and possibly M2 receptors that presumably contribute to prepulse inhibition.