Isolated segments of rat and guinea pig midcolon were used to examine the neurotransmitters responsible for ascending contraction and descending relaxation components of the peristaltic reflex. Graded radial stretch of the extreme orad end caused only descending relaxation accompanied by significant release of vasoactive intestinal peptide (VIP) in rat (82%, P less than 0.005) and guinea pig (47%, P less than 0.05). Radial stretch of the caudad end caused only ascending contraction without VIP release. VIP antiserum (1:480 to 1:60) inhibited descending relaxation in a concentration-dependent manner at all grades of stretch (40 +/- 12% to 74 +/- 15%) but augmented ascending contraction (25 +/- 7% to 108 +/- 21%). Axonal blockade with tetrodotoxin and ganglionic blockade with hexamethonium abolished both components, indicating the participation of cholinergic neurons. Atropine and the tachykinin antagonist [D-Pro2,D-Trp7,9]substance P inhibited ascending contraction but not descending relaxation; their combination abolished ascending contraction at all grades of stretch. We conclude that cholinergic neurons coupled to VIP motor neurons regulate descending relaxation and that cholinergic neurons coupled to tachykinin and cholinergic motor neurons regulate ascending contraction.