Acetylcholine is present in a majority of submucosal neurons which project to the intestinal epithelium. In this study, we examined the role of acetylcholine and the actions of cholinomimetic drugs, such as carbachol (CCH), on ion transport across muscle-stripped sheets of mucosa-submucosa from the proximal jejunum of weaned piglets. Serosal administration of CCH (10 nM-100 microM) produced rapid increases in short-circuit current (Isc) which were attributed to net Cl secretion. Acetylcholine, bethanechol and (4-hydroxy-2-butynyl)-1-trimethylammonium m-chlorocarbanilate chloride were partially effective in increasing Isc. Atropine and selective muscarinic cholinergic antagonists produced dextral shifts in the CCH concentration-effect relationship with an order of relative potency of 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP) greater than atropine much greater than pirenzepine greater than 11-[[[2-(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one(AF-DX116). The muscarinic receptor blocker [3H]quinuclidinyl benzilate (QNB) bound specifically and saturably to two sites in the mucosa-submucosa having equilibrium dissociation constants of approximately 10 +/- 3 and 890 +/- 120 pM and Bmax = 7 +/- 3 and 47 +/- 9 fmol/mg protein, respectively. Selective cholinergic antagonists competed for [3H]QNB binding with a rank order of affinity of 4-DAMP greater than hexahydrosiladifenidol much greater than AF-DX 116 greater than or equal to pirenzepine. Specific [3H]QNB binding sites were autoradiographically localized in the jejunal wall to the epithelium, submucosa, and muscularis propria. Electrical transmural stimulation (10-300 pulses/10 sec, 0.5 msec duration, 60 V stimulus strength) delivered to mucosal sheets produced tetrodotoxin-sensitive Isc elevations which were proportional to the number of impulses delivered. Mucosal Isc responses to electrical stimulation were attenuated by 10 microM hexamethonium, 1 microM atropine or autotachyphylaxis to CCH. Tetrodotoxin, at 0.1 microM, produced a 20-fold increase in the secretory potency of CCH. These results suggest that acetylcholine released from intramural neurons in porcine proximal jejunum produces transepithelial Cl secretion. Its effects may be mediated through interactions with two populations of muscarinic cholinergic receptors, located on neuronal and non-neuronal cells within the intestinal mucosa and submucosa, which serve to inhibit and promote Cl secretion respectively.