Headaches, especially migraine, involve not only pain but also aspects such as vasodilation of cranial vessels and sensitization of nerve endings, processes dependent on and connected to the central nervous system. To understand pathogenic mechanisms of headache, it is important to elucidate the central projections of sensory nerves that innervate cranial vessels, of which the middle meningeal artery (MMA) is the largest artery supplying the dura mater. In this study, cholera toxin subunit b (CTb) or wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) was applied on the adventitia of MMA. After perfusion fixation, the brainstem, the C1-C4 spinal segments and the trigeminal and C2 dorsal root ganglia were removed and sections from these tissues were processed to visualize transported tracers. Labeled cell bodies were seen ipsilaterally in the trigeminal and C2 dorsal root ganglia. Labeled nerve terminations were found ipsilaterally in the lateral part of the spinal dorsal horn of segments C1-C3 and in the caudal and interpolar parts of the spinal trigeminal nucleus. WGA-HRP labeled terminations were mainly located in laminae I and II, whereas CTb labeled terminations located in laminae III-V. These results indicate that sensory information from the MMA is transmitted through both trigeminal and cervical spinal nerve branches to a region in the central nervous system extending rostrally from the C3 dorsal horn to the interpolar part of the spinal trigeminal nucleus. Our data further substantiates that the sensory innervation of the MMA, in addition to putative nociceptive afferents, include a population of large caliber afferents with an as yet unclear but presumably non-nociceptive role.