The enteric nervous system (ENS) contains glutamatergic neurons, transporters and functional ionotropic and group I and II metabotropic glutamate receptors (mGluRs). The aim of this study was to determine whether the ENS contains functional group III mGluRs. RT-PCR demonstrated the expression of mGluR7 and mGluR8 mRNA in rat myenteric ganglia. Western blot analysis confirmed the presence of mGluR8 protein. Immunocytochemisty, in conjunction with confocal microscopy, demonstrated mGluR8 immunoreactivity in the ENS of several species, including humans. mGluR8 immunoreactivity was localized to the membrane of nerve cell bodies that received glutamatergic input. Significant receptor internalization of mGluR8 was observed upon activation and localization to membrane was observed upon blocking with the mGluR III antagonist (RS)-cyclopropyl-4-phosphonophenylglycine (CPPG). mGlu8-positive myenteric neurons contained glutamate or nitric oxide synthase (NOS), a marker of inhibitory motorneurons. Enteric group III mGluRs are functional because mGluR8 agonists inhibited forskolin-induced accumulation of cAMP in isolated myenteric ganglia, and CPPG reduced this effect. In addition, an accelerating effect on guinea pig colonic motility was observed following the application of mGluR8 agonists. The increase in motility was specific, since CPPG inhibited it. Moreover, in the presence of hexamethonium or N-nitro-L-arginine methyl ester, an inhibitor of NOS, responses caused by mGluR8 agonists were abolished. mGluR8 agonists also increased longitudinal muscle contractions. These findings suggest that mGluR8 agonists increase motility by inhibiting nitrergic relaxation and possibly, by facilitating cholinergic contractions.