Conventional and immunocytochemical, light- and electron-microscopic studies on the innervation of the pineal gland of the tree shrew (Tupaia glis) were made. Neuropeptide Y (NPY)-immunoreactive fibers, which were abundantly distributed in the gland, disappeared almost completely after superior cervical ganglionectomy, suggesting that these fibers are mostly postganglionic sympathetic fibers. By contrast, tyrosine hydroxylase (TH)-immunoreactive fibers, which were less numerous than NPY-fibers, remained in considerable numbers in ganglionectomized animals, indicating the innervation of TH-positive fibers from extrasympathetic sources. Bundles of substance P (SP)- or calcitonin gene-related peptide (CGRP)-immunoreactive fibers, entering the gland at its distal end, were left intact after ganglionectomy. SP-fibers were numerous, but CGRP-fibers were scarce in the gland. SP-immunoreactive fibers were myelinated and nonmyelinated, and were regarded as peripheral fibers because of the presence of a Schwann cell sheath. NPY- and SP-immunoreactive fibers and endings were mainly localized in the pineal parenchyma. NPY-immunoreactive endings synapsed frequently, and SP-positive ones did less frequently, with the cell bodies of pinealocytes. The results suggest that NPY and SP directly control the activity of pinealocytes. Sections stained for myelin showed that thick and less thick bundles of myelinated fibers entered the gland by way of the habenular and posterior commissures, respectively. Under the electron microscope, the bundles were found to contain also unmyelinated fibers. A considerable number of nerve endings synapsing with the cell bodies of pinealocytes remained in ganglionectomized animals; these endings were not immunoreactive for TH or SP. Such synaptic endings may be the terminals of commissural fibers.