Dysfunction of the hypocretin/orexin (Hcrt/Orx) peptide system is closely linked to the sleep disorder narcolepsy, suggesting that it is also central to the normal regulation of sleep and wakefulness. Indeed, Hcrt/Orx peptides produce long-lasting excitation of arousal-related neurons, including those in the laterodorsal tegmentum (LDT) and the dorsal raphe (DR), although the mechanisms underlying these actions are not understood. Since Hcrt/Orx mobilizes intracellular calcium ([Ca²⁺]_i) in cells transfected with orexin receptors and since receptor-mediated Ca²⁺transients are ubiquitous signaling mechanisms, we investigated whether Hcrt/Orx regulates [Ca²⁺]_iin the LDT and DR. Changes in [Ca²⁺]_iwere monitored by fluorescence changes of fura-2 AM loaded cells in young mouse brain slices. We found Hcrt/Orx (Orexin-A, 30-1,000 nM) evoked long-lasting increases in [Ca²⁺]_iwith differing temporal profiles ranging from spiking to smooth plateaus. A fragment of Hcrt/Orx (16-33) failed to evoke changes in [Ca²⁺]_iand changes were not blocked by TTX or ionotropic glutamate receptor antagonists, suggesting they resulted from specific activation of postsynaptic orexin receptors. Unlike orexin receptor–transfected cells, Hcrt/Orx-responses were not attenuated by depletion of Ca²⁺stores with cyclopiazonic acid (CPA; 3-30 μM), thapsigargin (3 μM), or ryanodine (20 μM), although store-depletion by either CPA or ryanodine blocked Ca²⁺mobilization by the metabotropic glutamate receptor agonist (±)-1-aminocyclopentane- trans-1,3-dicarboxylic acid ( trans-ACPD; 30 μM). In contrast, Hcrt/Orx responses were strongly attenuated by lowering extracellular Ca²⁺(∼20 μM) but were not inhibited by concentrations of KB-R7943 (10 μM) selective for blockade of sodium/calcium exchange. Nifedipine (10 μM), inhibited Hcrt/Orx responses but was more effective at abolishing spiking than plateau responses. Bay K 8644 (5-10 μM), an L-type calcium channel agonist, potentiated responses. Finally, responses were attenuated by inhibitors of protein kinase C (PKC) but not by inhibitors of adenylyl cyclase. Collectively, our findings indicate that Hcrt/Orx signaling in the reticular activating system involves elevation of [Ca²⁺]_iby a PKC-involved influx of Ca²⁺across the plasma membrane, in part, via L-type calcium channels. Thus the physiological release of Hcrt/Orx may help regulate Ca²⁺-dependent processes such as gene expression and NO production in the LDT and DR in relation with behavioral state. Accordingly, the loss of Hcrt/Orx signaling in narcolepsy would be expected to disrupt calcium-dependent processes in these and other target structures.