The effect of altering secretion into the Eustachian tube by modulating cholinergic innervation was studied in the anaesthetized rat. Active properties of the Eustachian tube were determined by measuring the ability of reflex-induced swallowing to equalize against an increased pressure level in the bulla. Reflex-induced swallowing was initiated by electrically stimulating the superior laryngeal nerve. Passive properties of the Eustachian tube were determined by increasing middle ear pressure until the Eustachian tube spontaneously opened. Blocking cholinergic neurotransmission with atropine had no effect on active or passive properties of the Eustachian tube. Potentiating cholinergic neurotransmission with neostigmine significantly impaired the ability of active swallowing to equilibrate middle ear pressure, but had no effect on passive properties of the Eustachian tube. The findings show that cholinergic nerve transmission, most likely from the parasympathetic division of the autonomic nervous system, can influence Eustachian tube function. We hypothesize that this effect is due to changes in surface tension in the Eustachian tube as a result of changes in secretion.