Opioid neurons exert a tonic restraint on inhibitory VIP/PACAP/NOS motoneurons of the enteric nervous system. A decrease in opioid peptide release during the descending phase of the peristaltic reflex, which underlies propulsive activity, leads to an increase in vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), and nitric oxide (NO) release and circular muscle relaxation. These effects are accentuated by opioid receptor antagonists. Endogenous opioid peptides and selective opioid -, - and µ-receptor agonists decreased the velocity of pellet propulsion in isolated segments of guinea pig colon, whereas selective antagonists increased velocity in a concentration-dependent fashion with an order of potency indicating preferential involvement of -receptors. 5-HT₄ agonists (HTF-919 and R-093877), which also increase the velocity of propulsion, acted synergistically with the -receptor antagonist naltrindole; a threshold concentration of naltrindole (10 nM) shifted the concentration-response curve to HTF-919 to the left by 70-fold. A combination of 10 nM naltrindole with threshold concentrations of the 5-HT₄ agonists caused significant increases in the velocity of propulsion (50 ± 7 to 77 ± 8%). We conclude that 5-HT₄ agonists and opioid -receptor antagonists act synergistically to facilitate propulsive activity in isolated colonic segments.