This study examined whether mucosal stimulation activates long secretomotor neural reflexes, and if so, to determine their organization. The submucosa of in vitro full thickness guinea pig ileal preparations was exposed in the distal portion and intracellular recordings were obtained from electrophysiologically identified secretomotor neurons. Axons in the intact mucosa of the oral segment were stimulated using a large bipolar stimulating electrode. In control preparations, a single stimulus pulse evoked a fast excitatory post-synaptic potential (EPSP) in 86% of neurons located 0.7-1.0 cm anal to the stimulus site. A stimulus train evoked multiple fast EPSPs but slow EPSPs were not observed. To examine whether mucosal stimulation specifically activated mucosal sensory nerve terminals, the mucosa/submucosa was severed from the underlying layers and repositioned. In these preparations, fast EPSPs could not be elicited in 89% of cells. Superfusion phorbol dibutyrate which enhances excitability of sensory neurons or pressure-pulse application of serotonin to the mucosa increased the fast EPSPs evoked by mucosal stimulation, providing further evidence that sensory neurons were involved. To determine if these reflexes projected through the myenteric plexus, this plexus was surgically lesioned between the stimulus site and the impaled neuron. No fast EPSPs were recorded in these preparations following mucosal stimulation whereas lesioning the submucosal plexus had no effect. These results demonstrate that mucosal stimulation triggers a long myenteric pathway that activates submucosal secretomotor neurons. This pathway projects in parallel with motor and vasodilator reflexes and this common pathway may enable co-ordination of intestinal secretion, blood flow and motility.