Orexin-A and orexin-B are hypothalamic neuropeptides that play critical roles in the maintenance of wakefulness. Intracerebroventricular (ICV) administration of orexin-A has been shown to promote wakefulness and suppress both rapid eye movement (REM) sleep and non-REM (NREM) sleep through the orexin receptor-1 (OX₁R) and orexin receptor-2 (OX₂R). Here, we elucidated the differential roles of orexin receptors in the regulation of sleep and wakefulness by comparing the effects of ICV orexin-A administration in wild-type, OX₁R^−/−, and OX₂R^−/− mice. The effects of orexin-A on wakefulness and NREM sleep were significantly attenuated in both knock-out mice as compared with wild-type mice, with substantially larger attenuation in OX₂R^−/− mice than in OX₁R^−/− mice. These results suggest that although the OX₂R-mediated pathway has a pivotal role in the promotion of wakefulness, OX₁R also plays additional roles in promoting arousal. In contrast, suppression of REM sleep by orexin-A administration was slightly and similarly attenuated in both OX₁R^−/− and OX₂R^−/− mice, suggesting a comparable contribution of the two receptors to REM sleep suppression. Histological studies demonstrated differential distributions of each receptor subtype in distinct neuronal populations with specific neurotransmitter identities in brainstem cholinergic/monoaminergic neurons. In the laterodorsal tegmental and pedunculopontine tegmental nuclei especially, cholinergic neurons exclusively expressed OX₁R mRNA, but OX₂R mRNA was expressed mainly in GABAergic putative interneurons. Thus, each orexin receptor subtype plays differential roles in gating NREM and REM sleep through distinct neuronal pathways.