The two essential regulating mechanisms of the middle ear pressure are the trans-mucosal gas exchange in the middle ear and the ventilation function of the eustachian tube (ET). The physiological mechanism of these both functions is not yet clear. The purpose of this study was to evaluate the role of the ET pressure equilibrium function by ventilation scintigraphy with technetium-99m. The rabbit animal model in vivo was used to study the presence and role of the ventilation of the tympanic cavity via auditory tube. The obtained results did not show any ventilation function of the ET despite active opening by muscle movement. In our experience, ventilation scintigraphy with technetium-99m is not a reliable method to study the auditory tube pressure equilibrium function in physiological conditions.