Background:Cannabinoid 1 (CB₁) receptors on myenteric neurons are involved in the regulation of intestinal motility. Aim: Our aim was to investigate CB₁ receptor involvement in ascending neurotransmission in mouse colon and to characterize the involved structures by functional and morphological means. Methods: Presence of the CB₁ receptor was investigated by RT-PCR and IHC was used for co-labeling studies. Myenteric reflex responses were initiated by electrical stimulation (ES) at different distances and junction potentials (JP) were recorded from circular smooth muscle cells by intracellular recording in a not-partitioned and a partitioned recording chamber. In vivo colonic propulsion was tested in wild-type and CB₁^-/- mice. Results: Immunostaining with the cytoskeletal marker, peripherin, showed CB1 immunoreactivity both on Dogiel type I and type II neurons. Further neurochemical characterization revealed CB₁ on ChAT-, calretinin- and 5-HT-immunopositive myenteric neurons. ES elicited neuronally mediated EJP (excitatory JP) and IJP (inhibitory JP). Gradual increases in distance resulted in a wave-like EJP with EJP amplitudes being maximal at the location of stimulating electrode (SE) 6 and a maximal EJP projection distance of ~18 mm. The CB₁ receptor agonist WIN 55,212-2 reduced the amplitude of EJP and was responsible for shortening the oral spreading of the excitatory impulse. In a partitioned chamber WIN 55,212-2 reduced EJP at the separated oral sites, proving that CB₁ activation inhibits interneuron mediated neurotransmission. These effects were absent in presence of the CB₁ antagonist SR141716A, which, when given alone, had no effect. WIN 55,212-2 inhibited colonic propulsion in wild-type mice but not in SR141716A-pretreated wild-type or CB₁^-/- mice. Conclusions: Activation of the CB₁ receptor modulates excitatory cholinergic neurotransmission in mouse colon by reducing amplitude and spatial spreading of the ascending electrophysiological impulses. This involves CB1 mediated effects on motor neurons and ascending interneurons and is likely to underlie the in vivo reduction in colonic propulsion.