The genesis of lower esophageal sphincter (LES) pressure in anesthetized opossums is a myogenic phenomenon without excitatory neural input. The mechanism responsible for generating phasic LES pressure phenomena in unanesthetized opossums, however, is not established. In this study, we evaluated the effects of potential pharmacological antagonists on LES pressure phenomena in unanesthetized opossums. We also compared LES responses to pharmacological excitatory agonists in anesthetized and unanesthetized animals. In awake animals the LES exhibited substantial tone as well as characteristic phasic activity. The tonic, or basal, LES pressure did not change during cycling of the migratory motor complex (MMC). However, atropine, 4‐diphenylacetoxy‐N‐methylpiperidine methiodide, and hexamethonium abolished the phasic, MMC‐related LES contractions while having no effect on basal pressure. Pirenzepine, prazosin, propranolol, pyrdamine, naloxone, and haloperidol did not affect either phasic or tonic LES pressure phenomena. Anesthesia substantially reduced the excitatory LES response to motilin but not to bethanechol, cholecystokinin‐octapeptide, pentagastrin, or phenylephrine. The results suggest that phasic LES contractions related to the MMC cycle are mediated by postganglionic cholinergic nerves with nicotinic ganglionic transmission. Basal LES tone, however, is maintained by a myogenic phenomenon. Anesthesia reduces excitatory LES responses induced by motilin, which acts via excitatory nerves, but has no effect on excitatory responses induced by agents that act mainly or exclusively on LES smoo