The concentration of acetylcholine (ACh) in rat jejunum that had been homogenized with an ultra-high-speed homogenizer (Biotron) was significantly higher than that in jejunum homogenized with a glass homogenizer. Rats were injected once or repeatedly for 10 days with a muscarinic agonist, pilocarpine (1 mg/kg), or a muscarinic antagonist, scopolamine (5 mg/kg). Animals were killed 20 min or 24 hr after single or consecutive injections, respectively, for determinations of cholinergic activities in the jejunum. Single treatment: Pilocarpine did not cause significant changes in the level of ACh, the activity of acetylcholinesterase (AChE), the binding of [3H]quinuclidinyl benzilate (QNB) or the contractile responses to ACh. Scopolamine reduced the level of ACh and binding of [3H]QNB without inducing significant changes in the activity of AChE and the contractile response. Consecutive treatment: Pilocarpine reduced the binding of [3H]QNB by changing the value of Bmax and reduced the contractile response without affecting the level of ACh or the activity of AChE. Scopolamine increased the binding of [3H]QNB without any effects on the level of ACh, the activity of AChE or the contractile response. In summary, it is possible to determine the level of ACh in a tissue as hard as intestine by homogenization with a Biotron and to assess the cholinergic situation in the intestine of animals that have been poisoned with various agents by estimating cholinergic activities.