Transport into the presynaptic terminal by the dopamine transporter is the primary mechanism for removing dopamine from the synaptic cleft. This transporter is a specific marker for dopamine terminals and is a primary site for CNS actions of cocaine. Several radioligands have been developed for analysis of the dopamine transporter. The ligands vary in affinity and specificity, leading to differences in reported transporter density in brain regions. We compared two of the most commonly used ligands, [3H]WIN 35,428 and [125I]RTI-55, analyzing the localization and density of sites in the rat brain using serial sections and quantitative autoradiography. Citalopram at 50 nmol/l was used to block [125I]RTI-55 binding to serotonin transport sites. Transporter density was highest in the striatum and both ligands labeled equivalent numbers of sites, with lateral to medial and anterior to posterior gradients. In most areas the density of sites measured with the two ligands was similar. However, [125I]RTI-55 binding was significantly higher than [3H]WIN 35,428 binding in the substantia nigra zona compacta, ventral tegmental area, subthalamic nucleus and a number of other subcortical nuclear groups while [3H]WIN 35,428 binding was higher in lateral striatum and in olfactory tubercle. These differences could reflect different forms of the transporter, perhaps due to post-translational modifications, and they may provide a basis for differential pharmacological regulation of transporter function in discrete brain regions and disease states.