Isolated segments of the guinea pig small intestine were used to examine the transmitter circuitry of the neural pathways subserving the ascending enteric reflex (AER) contraction of the circular muscle. Inflation of an intraluminal balloon provided the distension stimulus for the AER. The ascending contraction was reduced to 5% of its original amplitude by atropine and to 10% by hexamethonium, which indicates that cholinergic interneurons and cholinergic motor neurons constitute the main AER pathway. However, in the continued presence of atropine or hexamethonium for 60 min, the AER recovered to approximately 30% of its original amplitude. The atropine-resistant AER was blocked by hexamethonium and the tachykinin antagonist spantide [( D-Arg1,D-Trp7,9, Leu11]-substance P) suggesting that it involved cholinergic interneurons and tachykinin-utilizing motor neurons. The hexamethonium-resistant AER was abolished by atropine but left unaffected by spantide, suggesting the participation of as yet unidentified interneurons and cholinergic motor neurons. These findings demonstrate that the AER is mediated by multiple neural pathways with different transmitters and that adaptive interactions between these pathways take place after blockade of one of its neurotransmitters systems.