Endogenous opioids and opiate drugs have potent effects on intestinal function through activation of μ, δ and κ opioid receptors. Although the role of the μ receptor in these effects and its distribution within the gut wall has been extensively studied, little is known about the expression and function of the δ opioid receptor (DOR) in the intestine. Since the specificity of available DOR antibodies has been questioned (Cell 137:1-12, 2009), we have used an alternative, genetic strategy to determine the distribution and function of DOR in the enteric nervous system (ENS). Methods and Results. Mice in which enhanced GFP (eGFP) was knocked into the opioid delta receptor gene Oprd1 were studied. Western blotting for GFP revealed a protein of ~80kDa in all regions of the gut, consistent with the expected size of DOReGFP. DOReGFP was localized to myenteric and submucosal ganglia and nerve fibers within the muscularis externa and crypts by immunofluorescence, but not in enterocytes, smooth muscle and ICC. Although DOReGFP-positive neurons were rarely observed in submucosal ganglia, DOReGFP was expressed by ~50% of all PGP9.5-immunoreactive (IR) myenteric neurons throughout the intestine. DOReGFP was detected in both ChATand NOS-IR neurons of the duodenum (41%, 44%, respectively) and ileum (44%, 53%). DOReGFP also colocalized with ChAT in the cecum (30%), and proximal (9%) and distal colon (20%), but most DOReGFP-positive neurons in the large intestine coexpressed NOS-IR (66%, 96%, 65%, respectively). DOReGFP was not coexpressed with NFM in any region. DOReGFP function was investigated by examining agonist-stimulated trafficking of the receptor in myenteric wholemounts. In unstimulated preparations, DOReGFP was detected at the plasma membrane of the soma and neurites. The DOR agonists SNC80 (10, 100 nM) and met-enkephalin (100 nM) induced receptor clustering within 10 min and receptor trafficking to endosomes after 30 min. After 120-240 min, DOReGFP was in lysosomes, identified by LAMP1-IR. Equivalent results were obtained by live imaging of cultured neurons, and after administration of SNC80 to intact mice. SNC80 (100 nM) stimulated contraction of the isolated distal colon, confirming function. Conclusion. By using a novel approach for specific detection, we found that DOR is similarly distributed in cholinergic excitatory neurons and NOS containing inhibitory motor or interneurons in the small intestine, and that DOR is mostly confined to NOS-positive neurons of the large intestine. Activation induces DOR endocytosis and trafficking to lysosomes, where degradation will down-regulate opioid signaling. Supported by DK399857, NHMRC454858.