The μ-opioid receptor (μOR), which mediates many of the opioid effects in the nervous system, is expressed by enteric neurons. The aims of this study were to determine whether 1) different classes of myenteric neurons in the guinea pig ileum contain μOR immunoreactivity by using double- and triple-labeling immunofluorescence and confocal microscopy, 2) μOR immunoreactivity is localized to enteric neurons immunoreactive for the endogenous opioid enkephalin, and 3) μOR immunoreactivity is localized to interstitial cells of Cajal visualized by c-kit. In the myenteric plexus, 50% of μOR-immunoreactive neurons contained choline acetyltransferase (ChAT) immunoreactivity, whereas about 43% of ChAT-immunoreactive neurons were μOR immunoreactive. Approximately 46% of μOR myenteric neurons were immunoreactive for vasoactive intestinal polypeptide (VIP), and about 31% were immunoreactive for nitric oxide synthase (NOS). μOR immunoreactivity was found in about 68% of VIP-containing neurons and 60% of NOS-immunoreactive neurons. Triple labeling showed that about 32% of μOR neurons contained VIP and ChAT immunoreactivities. The endogenous opioid enkephalin (ENK) was observed in about 30% of μOR neurons; conversely, 48% of ENK neurons contained μOR immunoreactivity. μOR was not detected in neurons containing calbindin, nor in interstitial cells of Cajal. μOR-immunoreactive fibers formed a dense network around interstitial cells of Cajal in the deep muscular plexus. This study demonstrates that μOR is expressed by neurochemically distinct classes of myenteric neurons that are likely to differ functionally, is colocalized with the endogenous opioid ENK, and is not expressed by interstitial cells of Cajal. J. Comp. Neurol. 458:404–411, 2003. © 2003 Wiley-Liss, Inc.