The tuberomammillary nucleus (TMN) is the major source of histaminergic innervation of the mammalian brain and is thought to play a major role in regulating wake-sleep states. We recently found that sleep-active neurons in the ventrolateral preoptic nucleus (VLPO) provide a major input to the TMN, but the specificity of this projection and the neurotransmitters involved remain unknown. In this study, we examined the relationship of VLPO efferents to the TMN using both retrograde and anterograde tracing, combined with immunocytochemistry. We found that the descending projection from the VLPO selectively targets the cell bodies and proximal dendrites of the histaminergic TMN. In addition, VLPO axons could be traced into the brainstem, where they provided terminals in the the serotoninergic dorsal and median raphe nuclei, and the core of the noradrenergic locus coeruleus. Approximately 80% of the VLPO neurons that were retrogradely labeled by tracer injections including the TMN were immunoreactive either for galanin or for glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA. Virtually all of the galaninergic neurons in the VLPO were also GAD positive. Our results indicate that the VLPO may provide inhibitory GABAergic and galaninergic inputs to the cell bodies and proximal dendrites of the TMN and other components of the ascending monoaminergic arousal system. Because these cell groups are simultaneously inhibited during sleep, the VLPO sleep-active neurons may play a key role in silencing the ascending monoaminergic arousal system during sleep.