Recent evidence obtained from in vitro experiments indicates that cannabinoid receptor type 1 (CB1) modulates neurotransmission in the gastrointestinal tract of rodents. Standard intracellular recording techniques were applied to study the effects of cannabinoid receptor agonists and antagonists on smooth muscle resting membrane potentials and on membrane potentials following electrical neuronal stimulation in a myenteric neuron/smooth muscle preparation of wild-type and CB1-deficient mice in vitro. Double staining for CB1 and nitric oxide synthase (nNOS) was performed to identify the myenteric CB1-expressing neurons. Focal electrical stimulation of the myenteric plexus induced a fast excitatory junction potential (EJP; abolished by atropine, an inhibitor of cholinergic transmission) followed by a fast inhibitory junction potential (fIJP; reduced by apamin, a K⁺ channel blocker) and a slow inhibitory junction potential (sIJP; abolished by L-N(G)-nitroarginine, a NO synthase inhibitor). Treatment of wild-type mice with the endogenous CB1 receptor agonist anandamide significantly reduced EJP, while not affecting fIJP and sIJP. EJP was significantly higher in CB1-deficient mice than in wild-type littermate controls, and anandamide induced no effects in CB1-deficient mice. WIN 55,212-2, a synthetic CB1 receptor agonist, nearly abolished EJP and significantly reduced the fIJP in wild-type mice, while not affecting sIJP. SR141716A, a CB1 specific receptor antagonist, was able to reverse all the effects induced in wild-type mice by anandamide and WIN 55,212-2. SR141716A, when given alone, significantly increased EJP in wild-type mice without affecting IJP in wild-type and EJP in CB1-deficient mice. Interestingly, SR141716A reduced fIJP in CB1-deficient mice but not in wild-type mice. In the mouse colon, nitrergic myenteric neurons do not express CB1, implying that CB1 is expressed in cholinergic neurons. Excitatory and inhibitory neurotransmission in the mouse colon is modulated by activation of CB1 receptors. The significant increase in EJP in CB1-deficient mice strongly suggests a physiological involvement of CB1 in excitatory cholinergic neurotransmission. These electrophysiolgical findings suggesting the involvement of cholinergic neurons are in line with the immunocytochemical data indicating that CB1 receptors are expressed in non-nitrergic myenteric neurons. Finally, our study supports the notion that CB1 is the only receptor involved in the action of the endogenous agonist anandamide on the mouse proximal colon, since anandamide had no effect on excitatory or inhibitory neurotransmission in CB1-deficient mice.