The regulation mechanism of inhibition of intestinal ethanol absorption induced by high acetaldehyde (AcH) concentration in blood was investigated. We used atropine (AT), atropine methylbromide (ATMB), pirenzepine (PI), bethanechol (BE) and pilocarpine (PL) with or without cyanamide (CY; a potent inhibitor of aldehyde dehydrogenase, which induces high AcH concentration in blood). The K-a (absorption rate constant) value after the CY-alone pretreatment was significantly lower than that in controls. In the high AcH-induced cases, the values of K-a in AT and ATMB pretreatments were similar to controls, but the value of K-a in PI pretreatment was lower than that in controls. The values of K-a in the case of BE pretreatment with and without high AcH levels were lower than in controls. The K-a value in the PL with CY was significantly lower than that with CY alone. However, its action was blocked by ATMB pretreatment. These results suggest that high blood AcH concentrations inhibit intestinal ethanol absorption through the peripheral cholinergic nerves via muscarinic receptors, except for the muscarinic M-1 receptor, compared to other subtypes of muscarinic receptors.