Immunohistochemistry revealed the presence of tyrosine hydroxylase (TH)-, neuropeptide Y (NPY)-, calcitonin gene-related peptide (CGRP)- and substance P (SP)-immunoreactive nerve fibers and SP-immunoreactive neuronal cell bodies in the pineal gland of the cotton rat (Sigmodon hispidus). Abundant TH- and NPY-immunoreactive fibers were distributed evenly throughout the gland; less numerous CGRP- and SP-immunoreactive fibers were distributed in the superficial pineal and the stalk, but were scarce in the deep pineal. All the immunoreactive fibers were usually found around blood vessels. Since TH- and NPY-immunoreactive fibers in various pineal regions disappeared completely with superior cervical ganglionectomy, these fibers are all considered postganglionic sympathetic fibers. Intrapineal CGRP- or SP-immunoreactive fibers decreased considerably in number following superior cervical ganglionectomy, suggesting that some sympathetic fibers contain CGRP or SP. Bilateral bundles of nerve fibers under the transverse sinuses, corresponding to the nervi conarii, contained TH-, NPY-, CGRP- and SP-immunoreactive fibers, which continued into those distributed in the pineal capsule. In the nervi conarii, fibers immunoreactive for TH and NPY disappeared after superior cervical ganglionectomy, but those immunoreactive for CGRP and SP persisted. Thus, non-sympathetic, CGRP- and SP-immunoreactive fibers, together with sympathetic fibers, are presumed to enter the gland by way of the nervi conarii. Neuronal cell bodies, containing SP-like immunoreactivity and being possibly parasympathetic in nature, occurred occasionally in the superficial pineal.