The novel cocaine analog RTI-121[3β-(4-iodophenyl)tropane-2β-carboxylic acid isopropyl ester] was evaluated as a probe for the in vitro labeling and localization of the dopamine transporter in the human brain. Saturation binding experiments conducted in sucrose-phosphate buffer (10 mM sodium phosphate, pH 7.4, 0.32 M sucrose) revealed high- and low-affinity binding components with affinity values (KD) of 0.25 ± 0.04 and 4.9 ± 1.6 nM (mean ± SE) and densities (Bmax) of 56.8 ± 13.8 and 147.7 ± 23.4 pmol/g tissue, respectively. In contrast, when saturation binding experiments were performed in phosphate-buffered saline (10 mM Na2HP04, 1.8 mM KH2Po4, 136 mM NaCl,2.8 mM KC1, 10 mM NaI, pH 7.4), a 9-fold decrease in the density of the low-affinity component was noted, suggesting that the low-affinity RTI-121 binding site is associated with the region of the transporter involved in the ionic dependence of substrate recognition andor uptake. The rank order. of potency for inhibition of RTI-121 binding to human caudate membranes demonstrates that the radioligand selectively labels the dopamine transporter (GBR 12909 > RTI-121 > mazindol > nomifensine > (−)cocaine > desipramine > citalopram). Autoradiograpliic mapping of RTI-121 revealed very high densities of cocaine recognition sites over areas known to be rich in dopaminergic innervation, including the caudate, putamen, and nucleus accumbens. Moderate densities were also observed over the substantia nigra and the ventral tegmental area. Low-to-background labeling of RTI- 121 was seen throughout the cerebral cortex, amygdaloid nuclei, globus pallidus, and thalamus. In comparison with the autoradiographic distribution of the cocaine analogs L3H1WIN 35,428 (or CFT) and RTI-55 (or β-CIT), the labeling pattern for RTI-121 was more restricted. These studies demonstrate that RTI-121 labels dopamine-rich brain regions with greater selectivity than other currently available cocaine analogs, which makes it a potentially superior imaging probe for mapping the dopamine transporter in the human brain. © 1995 WiIey-Liss, Inc.