Background Aims Opioids and opiates inhibit gastrointestinal functions via I?, I , and I? receptors. Although agonists of the I opioid receptor (DOR) suppress motility and secretion, little is known about the localization and regulation of DOR in the gastrointestinal tract. Methods We studied mice in which the gene that encodes the enhanced green fluorescent protein (eGFP) was inserted into Oprd1, which encodes DOR, to express an approximately 80-kilodalton product (DOReGFP). We used these mice to localize DOR and to determine how agonists regulate the subcellular distribution of DOR. Results DOReGFP was expressed in all regions but was confined to enteric neurons and fibers within the muscularis externa. In the submucosal plexus, DOReGFP was detected in neuropeptide Ya??positive secretomotor and vasodilator neurons of the small intestine, but rarely was observed in the large bowel. In the myenteric plexus of the small intestine, DOReGFP was present in similar proportions of excitatory motoneurons and interneurons that expressed choline acetyltransferase and substance P, and in inhibitory motoneurons and interneurons that contained nitric oxide synthase. DOReGFP was present mostly in nitrergic myenteric neurons of colon. DOReGFP and I? opioid receptors often were co-expressed. DOReGFP-expressing neurons were associated with enkephalin-containing varicosities, and enkephalin-induced clathrin- and dynamin-mediated endocytosis and lysosomal trafficking of DOReGFP. DOReGFP replenishment at the plasma membrane was slow, requiring de novo synthesis, rather than recycling. Conclusions DOR localizes specifically to submucosal and myenteric neurons, which might account for the ability of DOR agonists to inhibit gastrointestinal secretion and motility. Sustained down-regulation of DOReGFP at the plasma membrane of activated neurons could induce long-lasting tolerance to DOR agonists.