The mammalian startle reflex is a fast response to sudden intense sensory stimuli that can be increased by anxiety or decreased by reward. The cellular integration of sensory and modulatory information takes place in giant neurones of the caudal pontine reticular formation (PnC). The startle reflex is known to be enhanced by 5-hydroxytryptamine (5-HT); however, signalling mechanisms that change the excitability of the PnC giant neurones are poorly understood. Possible molecular candidates are two-pore-domain K+ (K2P) channels that generate a variable K+ background conductance and control neuronal excitability upon activation of G-protein-coupled receptors. We demonstrate by in situ hybridization that the K2P channel TASK-3 is substantially expressed in PnC giant neurones. Brain slice recordings revealed a corresponding background K+ current in these cells that forms about 30% of the outward current at −30 mV. Inactivation of TASK-3 at pH 6.4 and by ruthenium red depolarized the cells by about 7 mV and increased the action potential frequency as well as duration. Specific activation of Gαq-coupled 5-HT2 receptors with α-methyl 5-HT evoked a similar increase of neuronal excitability. Consistently, we measured afferent synaptic inputs from serotonergic raphe neurones and detected 5-HT2C receptors in PnC giant neurones by immunohistochemistry. Thus, neuronal excitability of PnC giant neurones in vivo is most likely increased by serotonergic projections via the K2P channel TASK-3.