The participation of opioid neurons in the regulation of peristalsis was examined in a rat colonic segment that permits separate characterization of the components of the peristaltic reflex (ascending contraction and descending relaxation). Naloxone increased descending relaxation and decreased ascending contraction; opioid peptides [methionine-enkephalin (Met-Enk), dynorphin-13, and morphiceptin] had opposite effects. Naloxone increased, and Met-Enk decreased, vasoactive intestinal peptide (VIP) release during each component of the reflex. The changes in VIP release reinforced the direct effects of naloxone and opioid peptides on circular muscle tone, providing an explanation for the effects of these agents on the two components of the peristaltic reflex. Dynorphin release decreased during descending relaxation and increased during ascending contraction, reflecting corresponding changes in opioid neural activity. Based on these results a model is proposed, according to which a decrease in opioid neural activity during the initial phase (i.e., descending relaxation) results in direct and VIP-mediated decrease in circular muscle tone. Restoration of opioid neural activity during the subsequent phase (i.e., ascending contraction) increases circular muscle tone and reinforces the action of tachykinin and cholinergic motor neurons, which are the direct mediators of ascending contraction.