Intestinal motor activity associated with acetylcholine (ACh) release was assessed in the small intestine of anesthetized dogs by simultaneous measurement of motor activity and local ACh concentrations within the intestinal wall with in vivo microdialysis. Basal concentration of ACh measured in the dialysate was 1.12 +/- 0.08 pmol/15 min (n = 10), a value that remained constant until 3 h after perfusion. Intra-arterial infusion of tetrodotoxin reduced dialysate ACh concentration, while the motor activity accelerated at the early phase after infusion of tetrodotoxin and then decreased, thereby suggesting that the motor activity is regulated by not only excitatory cholinergic neurons, but also inhibitory neurons. Intraarterial infusion of atropine increased dialysate ACh concentration but reduced motor activity, thereby indicating that the cholinergic neurons are tonically active and the muscarinic autoreceptors operate to inhibit the ACh release. Intraarterial infusion of norepinephrine reduced, but yohimbine increased both motor activity and dialysate ACh concentration, thereby indicating that the adrenergic neurons regulate the motor activity due to control of cholinergic neuronal activity. This in vivo microdialysis method demonstrated in the whole body of animals that the activity of cholinergic neurons was physiologically regulated by itself and adrenergic neurons.