The peptidergic and serotoninergic innervation of the rat dura mater was investigated by reacting dural wholemounts immunohistochemically with antibodies to calcitonin gene-related peptide (CGRP), substance P (SP), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), and serotonin (5-HT). CGRP and SP innervations of the dura were robust and the patterns of distribution of these neuropeptides were essentially the same. The majority of the fibers were perivascular and distributed to branches of the anterior and middle meningeal arteries and to the superior sagittal and transverse sinuses. Other CGRP/SP fibers appeared to end "free" within the dural connective tissue. NPY-immunoreactive fibers were extremely numerous and also distributed heavily to the branches of the meningeal arteries, the venous sinuses, and to the dural connective tissue. The pattern of NPY innervation resembled in many ways that of CGRP/SP; however, NPY innervation of the sinuses was greater and NPY perivascular fibers supplying the meningeal arteries formed more intimate contacts with the walls of the vessels. The pattern of VIP innervation was, in general, similar to that observed for the three previous neuropeptides; however, the overall density was considerably less. Small to moderate numbers of serotoninergic nerve fibers were observed in some, but not all, of the duras processed for 5-HT. The latter fibers were almost exclusively perivascular in distribution. Dural mast cells were prominently stained in the 5-HT preparations because of their serotonin content. Mast cells were also labeled in a nonspecific fashion in some of the tissues reacted immunohistochemically for neuropeptides; some of them were located in close apposition to passing nerve fibers. This study represents, to our knowledge, the first comprehensive work on the peptidergic and serotoninergic innervation of the mammalian dura mater. The results should increase our understanding of the roles that these fibers play in normal dural physiology and of their potential interactions in the pathogenesis of vascular headache.