The endogenous opioid enkephalin drives ion transport towards absorption. To determine the site and mechanism of this effect, fractionated stripping of guinea-pig ileum was carried out. The muscularis propria, including myenteric plexus, was removed by partial stripping. The submucosa, including the submucosal plexus, plus the muscularis mucosae were removed by total stripping. For binding studies, epithelial cells were removed by the method of Weiser leaving the lamina propria mucosae with the mucosal plexus. Radio-receptor-assay with (3H)2-D-ala-5-D-leu-enkephalin revealed enkephalin binding sites in the submucosa plus muscularis mucosae (KD = 3.6 nmol l-1; Vmax = 7.3 fmol mg-1) and in the lamina propria mucosae (KD = 4.2 nmol l-1; Vmax = 5.1 fmol mg-1. The binding was stereospecific in both layers. No binding was detected on epithelial cells. In the Ussing chamber, partially stripped ileum exhibited spontaneous ISC which was abolished by addition of tetrodotoxin (TTX) or by total stripping indicating that this ISC was neuronally stimulated by the submucosal plexus. Electrogenic chloride secretion was identified as contributing to this ISC, since the TTX-sensitive part of ISC in the partially stripped ileum was lacking in Cl- and HCO3-free medium, reappeared after addition of Cl consistent with Michaelis-Menten kinetics (Km = 19 nmol l-1) and was reversed by serosal addition of bumetanide. In addition, enkephalin increased electroneutral NaCl-absorption as obtained by Na- and Cl-flux measurements. Enkephalin decreased this spontaneous neuronally stimulated electrogenic Cl-secretion in the partially stripped ileum, but had no effect in totally stripped ileum if ISC was stimulated at the cellular level by theophylline or PGE1. We conclude that ganglia located in the submucosal plexus regulate intestinal ion transport. Enkephalin acts by presynaptic inhibition via receptors on these neurons in the submucosa and/or via receptors on their neurites in the lamina propria mucosae.