The drug 3 beta-[4'-iodophenyl]tropan-2 beta-carboxylic acid methyl ester (RTI-55) is a cocaine congener with high affinity for the dopamine transporter (Kd < 1 nM). The present study characterized [125I]RTI-55 binding to membranes prepared from rat, monkey and human caudates and COS cells transiently expressing the cloned rat dopamine (DA) transporter. Using the method of binding surface analysis, two binding sites were resolved in rat caudate: a high-capacity binding site (site 1, Bmax = 11,900 fmol/mg of protein) and a low-capacity site (site 2, Bmax = 846 fmol/mg of protein). The Kd (or Ki) values of selected drugs at the two sites were as follows: (Ki for high-capacity site and Ki for low-capacity site, respectively): RTI-55 (0.76 and 0.21 nM), 1-[2-diphenyl-methoxy)ethyl]-4-(3-phenylpropyl)piperazine (0.79 and 358 nM), mazindol (37.6 and 631 nM), 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (45.0 and 540 nM) and cocaine (341 and 129 nM). Nisoxetine, a selective noradrenergic uptake blocker, had low affinity for both sites. Serotonergic uptake blockers had a high degree of selectivity and high affinity for the low-capacity binding site (Ki of citalopram = 0.38 nM; Ki of paroxetine = 0.033 nM). The i.c.v. administration of 5,7-dihydroxytryptamine to rats pretreated with nomifensine (to protect dopaminergic and noradrenergic nerve terminals) selectively decreased the Bmax of site 2, strongly supporting the idea that site 2 is a binding site on the serotonin (5-HT) transporter. This serotonergic lesion also increased the affinity of [125I]RTI-55 for the DA transporter by 10-fold. The ligand selectivity of the caudate 5-HT transporter was different from the [I125]RTI-55 binding site on the 5-HT transporter present in membranes prepared from whole rat brain minus caudate. The [125I]RTI-55 binding to the DA transporter was further resolved into two components, termed sites 1a and 1b, by using human and monkey (Macaca mulatta) caudate membranes but not the membranes prepared from rat caudate or COS cells that transiently expressed the cloned cocaine-sensitive DA transporter complementary DNA. Similar experiments also resolved two components of the caudate 5-HT transporter. Viewed collectively, these data provide evidence that [125I]RTI-55 labels multiple binding sites associated with the DA and 5-HT transporters.