The postsynaptic effects of acetylcholine (ACh) on excitatory neurones in the neocortex are generally believed to be predominantly excitatory. Indeed, neocortical pyramidal neurones are depolarised by ACh acting on M1 muscarinic receptors, even though a brief transient hyperpolarizing component may occur under certain conditions. Here we demonstrate that layer 4 (L4) spiny neurones of the primary somatosensory cortex are persistently and monophasically hyperpolarized by ACh acting via M4R muscarinic receptors. This hyperpolarisation, correlated with a decrease in membrane resistance, strongly increases the current threshold to evoke action potentials in L4 spiny neurons. In parallel, paired recordings of spiny neurons indicate that ACh causes a reduction in synaptic efficacy probably via a decrease in release probability. This combination of pre- and postsynaptic effects of ACh contributes to an increased signal-to-noise ratio in the recurrent L4 microcircuit during waking and sustained attention. Together with its hyperpolarising action, ACh decreases the slow afterhyperpolarisation (sAHP) following action potentials in L4 spiny neurones, thereby improving the temporal fidelity of their response properties. Finally, it appears that these effects of ACh may be a general feature of primary sensory cortices, since L4 spiny neurons of the visual (V1) and the auditory cortex (A1) are also persistently hyperpolarized by ACh.