Abstract: The cocaine analogue RTI-55 was evaluated as a probe for in vitro labeling and localization of dopamine and serotonin transporters after death in the human brain. Kinetic, saturation, and competition binding experiments indicated complex interactions of the radioligand with the identification of multiple recognition sites. In membrane binding assays, the association of [125I]RTI-55 at 25°C to putamen membranes was monophasic. In contrast, dissociation of [125I]RTI-55 occurred in two phases with t1/2 values of 9.4 and 36.5 min, respectively. Saturation analysis of [125I]RTI-55 binding demonstrated two binding sites in the human putamen with KD values of 0.10 ± 0.02 and 1.81 ± 0.46 nM. The binding of [125I]RTI-55 was displaced by a wide range of cocaine analogues and monoamine uptake inhibitors. The rank order of potency demonstrated in competition assays with human putamen membranes indicates that the radioligand labels cocaine recognition sites on the dopamine transporter (mazindol > GBR 12909 > GBR 12935 > paroxetine > nisoxetine > desipramine ≥ fluoxetine > citalopram). In the human occipital cortex, [125I]RTI-55 recognized multiple binding sites with KD values of 0.02 ± 0.01 and 4.18 ± 0.46 nM. The rank order of potency for inhibition of [125I]RTI-55 binding to cerebral cortex membranes (paroxetine > citalopram > GBR 12909 ≥ mazindol ≥ nisoxetine > benztropine) suggests that [125I]RTI-55 labels the serotonin transporter in the human occipital cortex. Autoradiographic mapping of [125I]RTI-55 revealed very high densities of cocaine recognition sites over areas known to be rich in dopaminergic innervation, including the caudate, putamen, and nucleus accumbens. Moderately elevated densities of [125I]RTI-55 binding sites were also seen throughout the thalamus, hypothalamus, and substantia nigra. [125I]RTI-55 binding sites were prevalent throughout the cerebral cortex and amygdala. In autoradiographic studies, the addition of the selective serotonin transport blocker citalopram completely prevented [125I]RTI-55 labeling in the thalamus, hypothalamus, and throughout most of the cerebral cortex. In the presence of citalopram, [125I]RTI-55 binding site densities remained elevated over the striatum and substantia nigra, with selective residual labeling also seen in the external segment of the globus pallidus and the lateral nucleus of the amygdala. These results demonstrate that in the human brain, [125I]RTI-55 labels multiple recognition sites on dopamine and serotonin transporters.