Lower esophageal sphincter pressures were monitored with water-filled catheters in anesthetized opossums. Muscarinic agonists McN-A-343 and bethanechol were administered in the arterial supply of the sphincter. McN-A-343 caused relaxation after a brief contraction of the sphincter. Bethanechol caused a dose-dependent contraction. Tetrodotoxin antagonized the inhibitory effect of McN-A-343 but did not antagonize sphincter contraction caused by McN-A-343 or bethanechol. The mean ED50 [effective dose to achieve 50% protection] values were 6.9 nmol/kg i.a. for McN-A-343-induced relaxation, 10.5 nmol/kg i.a. for McN-A-343-induced contraction and 0.4 nmol/kg i.a. for bethanechol-induced contraction. Atropine caused a dose-dependent rightward shift in the dose-response curves of inhibitory and excitatory effects of the 2-muscarinic agonists. Pirenzepine caused a dose-dependent rightward shift in the dose-response curves of McN-A-343-induced relaxation. Pirenzepine did not modify sphincter contraction caused by themuscarinic agonists. 4-Diphenylacetoxy-N-methylpiperidine methiodide did not modify McN-A-343-induced sphincter relaxation but caused dose-dependent rightward shifts in the dose-response curves of sphincter contraction caused by McN-A-343 or bethanechol. There are apparently 2 distinct types of muscarine receptors in the opossum lower esophageal sphincter. The M1 muscarine receptors are present on the inhibitory neurons and participate in the synaptic transmission between vagal preganglionic and intramural post-ganglionic inhibitory neurons. They are activated by McN-A-343 and antagonized by pirenzepine. The M2 muscarine receptors are located directly on the sphincter muscle. They are also activated by McN-A-343, but are selectively activated by benthanechol and are antagonized by 4-diphenylacetoxy-N-methylpiperidine methiodide.