The dopaminergic system has been proposed to be one of the innervations in controlling the mammalian pineal gland function. The dopamine receptors have been characterized in the pineal and the biphasic effects of dopamine on melatonin production have been demonstrated. Recently, the site of dopamine transporter (DAT), a plasma membrane transport protein of dopaminergic neuron, also has been characterized in the bovine pineal gland. The aim of the present study was to identify the dopaminergic innervation in the bovine pineal gland. The localization and expression of DAT have been performed by using an immunohistochemical method and a reverse transcriptase polymerase chain reaction (RT-PCR) technique. DAT-immunoreactivity was found in the nerve terminals throughout the gland, but not in pinealocytes or neuronal-like cells. Some DAT-immunoreactive nerve fibers were observed along the pineal stalk. DAT mRNA product from RT-PCR was found in the bovine substantia nigra, but not in the pineal gland. The colocalization of DAT with tyrosine hydroxylase (TH) or dopamine beta hydroxylase (DBH) immunoreactivities was observed in nerve terminals. However, no colocalization of DAT with DBH was found in some terminals/fibers. The present results showed the central dopaminergic innervation in the bovine pineal gland distinctively from noradrenergic nerve fibers, and their perikarya origin was located possibly outside of the gland.