An immunohistochemical investigation of the bovine pineal gland was performed using both a rabbit polyclonal antibody and a rat monoclonal antibody against choline acetyltransferase (ChAT). A network of ChAT-immunoreactive (IR) nerve fibers was located throughout the pineal gland, both in the perivascular spaces and between the pinealocytes. Most of the intrapineal ChAT-IR nerve fibers were endowed with varicosities. In addition, some ChAT-IR intrapineal neurons were found, often located at the base of the gland near the pineal recess. Within the habenular nucleus and pineal stalk, ChAT-IR perikarya and nerve fibers were also present. Some of these fibers projected towards the pineal gland. A number of ChAT-IR nerves were also located in the posterior commissure and could be followed into the gland. At the caudal tip of the pineal gland, a bundle of ChAT-IR nerve fibers was observed to penetrate into the gland together with blood vessels. The presence of a cholinergic innervation of the bovine pineal gland, together with previous demonstration of the presence of choline acetyltransferase and muscarinic receptor binding sites in the bovine pineal gland, indicates a functional influence of a cholinergic nervous system on the pinealocyte.