The cholinergic system plays an important role in regulation of arousal and REM sleep. In the present study, we showed that a muscarinic receptor agonist, carbachol (CCh), activates almost 20% of orexin-producing neurons (orexin neurons), which play a critical role in maintenance of arousal. We also found that a very small population of orexin neurons (1%) was inhibited by CCh. Muscarinic receptor antagonists inhibited the CCh-induced activation of orexin neurons in a dose-dependent manner. The M(3)-selective muscarinic receptor antagonist 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) affected the response at a significantly lower concentration compared with the M(1)-selective muscarinic antagonist pirenzepine. IC(50) of 4-DAMP and pirenzepine was 0.30 +/- 0.15 and 59.9 +/- 18.6 nM, respectively. The CCh-induced inward current was attenuated by the nonselective cation channel blocker SKF96365. These results indicate that CCh activates 20% of orexin neurons through the M(3) muscarinic receptor and subsequent activation of nonselective cation channels.