We have recently identified a population of mechanosensory myenteric S-interneurons in the distal colon of guinea-pigs. However, the role of the longitudinal (LM) and circular muscle (CM) in transducing these mechanosensory signals into enteric reflexes is unclear. In this study, we have investigated whether the LM or CM layer is necessary for activation of ascending excitatory and descending inhibitory neuronal pathways by static stretch of the paralysed isolated guinea-pig distal colon. Simultaneous intracellular recordings were made from pairs of CM cells at either end of isolated sheet preparations of distal colon that were devoid of mucosa and submucous plexus; and were maintained under circumferential stretch. In the presence of nifedipine (1 microm), an ongoing discharge of excitatory junction potentials (EJPs) and inhibitory junction potentials (IJPs) were recorded simultaneously at the oral and anal ends of the preparation. When the LM was sharp dissected off the myenteric plexus, the synchronized discharge of ascending EJPs and descending IJPs in the CM layer was unaffected. In contrast, when the majority of CM was sharp dissected off the myenteric plexus, ongoing neural activity was absent, or substantially decreased in both the LM and CM. In these preparations, immunohistochemical staining, together with transmural electrical stimuli confirmed that the myenteric plexus was always present and intact in these preparations. When full-thickness strips of CM were removed from progressively longer lengths of myenteric plexus, a graded reduction in the correlation of coordinated oral EJPs and anal IJPs occurred. However, removing approximately 40% of the thickness of CM layer from the entire preparation did not significantly disrupt, nor reduce the degree of correlation between oral EJPs and anal IJPs, suggesting that critical sensory elements did not lie adjacent to the submucosal plexus. It is concluded that mechanosensory transmission that underlies repetitive firing of ascending excitatory and descending inhibitory neuronal pathways is critically dependent upon sensory elements within the CM layer. These elements are likely to activate stretch-sensitive interneurons in the myenteric plexus. No evidence was found to suggest that the connectivity between the LM and the myenteric plexus was required for mechanotransduction.