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The mammalian pineal gland contains several neurotransmitters and receptors for amino acids, biogenic amines, and peptides. Some of these, such as D1 and D2 dopamine receptors, have been previously identified and characterized in the bovine pineal gland by our group. As a matter of fact, the density of D1 dopamine receptors in the pineal gland is higher than that of corpus striatum, suggesting that this organ must possess a high affinity dopamine transporter, which has been identified in this study by using [3H]GBR 12935 as a radiological ligand and nomifensine to determine non-specific binding. The association rate of [3H]GBR 12935 binding to the pineal membrane was examined as a function of time. The binding reached equilibrium within 45 min of incubation at 25°C. The specific binding was reversible and saturable. The dissociation time course of the specific [3H]GBR 12935 binding from the bovine pineal membrane was also studied. A half-life (t1/2) of 14-min was obtained. The saturation analysis of the [3H]GBR 12935 binding revealed a dissociation equilibrium constant (Kd) of 6.0 ± 0.9 nM and a receptor density (Bmax) of 6.9 ± 0.3 pmol/mg protein, which were comparable with those values obtained from bovine striatum and frontal cortex. In competitive experiments, the concentrations of drugs required to inhibit 50% of the binding (IC50) were in descending order GBR 12909 > GBR 12935 > trans-flupenthixol > nomifensine > cis-flupenthixol > amitriptyline > imipramine > desipramine > dopamine > fluoxetine > fuvoxamine > d-amphetamine. However, nisoxetine, SCH 23390, norepinephrine, and serotonin were unable to displace [3H]GBR binding. These results show that drugs capable of blocking dopamine transporters were effective in displacing [3H]GBR binding; whereas specific norepinephrine and serotonin transporter inhibitors were less effective or ineffective. In addition, the dopamine transporter is ion-dependent as sodium increased [3H]GBR binding in a concentration related manner. These results indicate that a high affinity dopamine transporter exists in the bovine pineal, which may exhibit circadian periodicity, and whose physiological functions need to be delineated and characterized in future investigations.