In vivo, thalamocortical axons are susceptible to the generation of terminal spikes which antidromically promote bursting in the thalamus. Although neurotransmitters could elicit such ectopic action potentials at thalamocortical boutons, this hypothesis has never been confirmed. Prefrontal cortex is the cortical area most implicated in arousal and is innervated by thalamic neurons that are unusual since they burst rhythmically during waking. We show that a neurotransmitter critical for alertness, hypocretin (orexin), directly excites prefrontal thalamocortical synapses in acute slice. This TTX-sensitive activation of thalamic axons was demonstrated electrophysiologically and by two-photon sampling of calcium transients at single spines in apposition to thalamic boutons anterogradely labeled in vivo. Spines receiving these long-range projections constituted a unique population in terms of the presynaptic excitatory action of hypocretin. By this mechanism, the hypocretin projection to prefrontal cortex may play a larger role in prefrontal or "executive" aspects of alertness and attention than previously anticipated.