Cannabis has been used for centuries in the medicinal treatment of gastrointestinal disorders. Endogenous cannabinimimetic substances such as 2-arachidonylglycerol have been isolated from gut homogenates and CB₁-cannabinoid binding sites have been identified in small intestine. In this study, CB₁-cannabinoid receptors (CB₁-R) were immunohistochemically localized within the enteric nervous system of the pig, an omnivorous species whose digestive tract is functionally similar to humans. Two anti-CB₁-R antisera, raised against N-terminal epitopes in the human CB₁-R, were employed to localize receptor immunoreactivity by secondary immunofluorescence. CB₁-R immunoreactivity was observed in the myenteric and submucosal ganglionated plexuses of porcine ileum and colon. In the ileum, all CB₁-R-immunoreactive neurons coexpressed immunoreactivity to the cholinergic marker, choline acetyltransferase (ChAT). CB₁-R/ChAT-immunoreactive neurons appeared to be in close apposition to ileal Peyer's patches, submucosal blood vessels, and intestinal crypts. In the distal colon, CB₁-R-immunoreactive neurons also expressed immunoreactivity to ChAT, albeit less frequently than in ileum. Immunoreactivity to vasoactive intestinal peptide or nitric oxide synthase was not colocalized in ileal or colonic CB₁-R-immunoreactive neurons. These studies indicate that CB₁-R are present in cholinergic neurons in the porcine enteric nervous system. The potential roles of these receptors in intestinal motility and epithelial transport, host defense and visceral pain transmission are discussed.