Using a new application of the endoluminal approach, we were able to demonstrate the sonographic anatomy of the eustachian tube in vitro and in vivo and correlate it with the plastic-embedded specimen. Five adult normocephalic cadavers, two patients, and one specimen especially prepared for embedding in plastic were examined. A specifically developed device was used to insert the ultrasound transducer employing an endo-oral approach. The investigation was performed using an intravascular ultrasound unit. In all cases the tube could be visualized in its entirety and relevant anatomic structures identified and compared with the specimen at the corresponding levels. The deeper layers, including the paratubal structures and the mucosa, could be distinguished for the first time by means of endoluminal ultrasonography. Now that endoluminal ultrasonography has revealed this anatomic information, further studies will be able to gauge the clinical efficacy of our method in cases of ventilatory, drainage, and clearance problems. The images showed no difference between the structures in vitro and in vivo, and the vivo examination was even easier than the in vitro one owing to better tissue turgor.