Activation of cannabinoid CB₁ receptors inhibits gastrointestinal motility, propulsion, and transit, whereas selective antagonism of these receptors has the opposite effects, suggesting the presence of endocannabinoid tone. Supporting evidence for presynaptic CB₁ receptors on myenteric neurons has been found in vitro. In this study, selective CB₁ receptor antibodies and neuronal markers were used to identify and characterise myenteric neurons expressing cannabinoid receptors. Whole mounts of rat and guinea pig myenteric preparations were dually labelled with antibodies against the CB₁ receptor and choline acetyltransferase, neurofilament proteins, calbindin, calretinin, synapsin I, microtubule‐associated protein‐2, calcitonin gene‐related peptide, or substance P. The pattern of CB₁ receptor labelling and the neurochemical classification of CB₁ receptor‐positive cells were markedly influenced by the species and fixation procedure. Virtually all choline acetyltransferase‐immunoreactive myenteric neurons expressed CB₁ receptors in ganglia from both species. Subpopulations of neurons identified with calbindin, calretinin, and microtubule‐associated protein‐2 did not express CB₁ receptors. A few calcitonin gene‐related peptide‐ and substance P‐positive somata coexpressed CB₁ receptor immunoreactivity but showed little colocalisation on individual fibres. There was a close association between CB₁ receptor immunoreactivity and fibres labelled for synaptic protein, suggesting a role in the modulation of transmitter release. Functional responses to cannabinoids in the presence of hexamethonium suggest further that CB₁ receptors occur on excitatory motoneurons. In conclusion, CB₁ receptors are expressed on a variety of cholinergic sensory, interneuronal, and motor neurons in myenteric ganglia. J. Comp. Neurol. 448:410–422, 2002. © 2002 Wiley‐Liss, Inc.