Objectives/HypothesisThe eustachian tube (ET) is an important landmark in skull base surgery, which has a close relationship with the petrous segment of the internal carotid artery (ICA). The goal of the current study was to establish the detailed anatomic relationship of the ET and petrous segment of the ICA. Study DesignAnatomical study. MethodsSix silicon-injected adult cadaveric heads (12 sides) were dissected using a lateral infratemporal fossa approach (type C) and endoscopic endonasal approach. The ET and ICA were exposed; their detailed relationships were demonstrated. High-quality pictures were obtained. ResultsIn the anterior genu/foramen lacerum segment of the ICA, the vidian nerve was an important landmark. The cartilaginous ET was divided into four segments, from anterior to posterior: nasopharyngeal, pterygoid, lacerum, and petrosal segment. The anterior and inferior wall of the carotid canal was consistently between the horizontal ICA and petrous segment of the cartilaginous ET. In the posterior genu of the ICA, the bony part of the ET, and the tendon of the tensor tympani muscle were paramount landmarks. The posterior genu of the ICA was imbedded in the carotid canal. The landmarks of the junction of the cartilaginous ET and bony ET were the sphenoid spine and foramen spinosum. ConclusionsThe anatomical segmentation of the ET provides the basis for safe and effective transection of the ET in skull base surgery. An understanding of the complex relationships of the ET and petrous segment of the ICA is paramount for surgically dealing with disease located within the region of the ET and petrous segment of the ICA. Level of EvidenceNA Laryngoscope, 124:2655-2664, 2014