The aims of the study were: 1. to evaluate the in vivo interaction of opioid and cholinergic receptors in the regulation of Met-enkephalins (native and total) and ghrelin concentrations in the rat intestine; 2. to explore the association of opioids, muscarinic and nicotinic receptors in the regulation of in vitro secretion of peptidergic hormones such as Met-enkephalin, ghrelin, gastrin and somatostatin from rat intestine. Two experiments were performed on adult, male Wistar rats: 1. in in vivo experiment animals received a single intraperitoneal injection of NaCl (control), naltrexone (opioid receptor antagonist), atropine (muscarinic receptor antagonist) or hexamethonium (nicotinic receptor antagonist), and Met-enkephalins concentration and expression of mRNA for proenkephalin, and ghrelin concentration were measured in the intestine; 2. in in vitro experiments above receptors antagonists were added to tissue culture alone or in combination; native Met-enkephalin, ghrelin, gastrin and somatostatin concentrations were measured in the medium. In vivo inhibition of receptors increased the concentration of total Met-enkephalin and potentiated the expression of mRNA for proenkephalin. Ghrelin concentration was increased by naltrexone and decreased by atropine. In vitro secretion of Met-enkephalin was decreased by both cholinergic receptor antagonists but this effect was fully reversed by naltrexone. Ghrelin and gastrin release from the intestine was increased by all three antagonists given alone; in vitro somatostatin release from the intestine was attenuated by receptor antagonists given alone or in combination. The obtained results clearly showed the interaction of opioids and acetylcholine in the regulation of synthesis, concentration and release of peptidergic hormones from the rat intestine.