Basal release of vasoactive intestinal polypeptide (VIP) was 100-fold higher than substance P (SP) release from the vascularly perfused, neurally isolated canine small intestine. High-frequency field stimulation increased SP release but decreased VIP release. VIP release was markedly reduced by tetrodotoxin, perfusion with Ca-free medium, or by hexamethonium but not by atropine. Acetylcholine increased VIP output by an atropine-sensitive mechanism. Methionine-enkephalin or dynorphin (at 10-fold higher concentrations) markedly reduced VIP output; the actions of both these were abolished by naloxone. BHT-920, an alpha 2-adrenoceptor agonist, reduced VIP output markedly by a rauwolscine-sensitive mechanism. Isoproterenol and phenylephrine were without effect. Motilin produced persistent inhibition of VIP output. Thus VIP neurons of isolated canine small intestine were continuously active, driven by intrinsic cholinergic nerves, and subject to presynaptic inhibition by opioid agonists, alpha 2-adrenoceptor agonists, and motilin. This intrinsic neural system may provide tonic inhibitory control to the small intestinal circular muscle and provide a mechanism by which agents may modulate intestinal motor function.