Previous studies showed that the cocaine analog [125I]RTI-55 labels dopamine and serotonergic (5-HT) biogenic amine transporters (BATs) with high affinity. Here we characterized [125I]RTI-55 binding to membranes prepared from whole rat brain minus the caudate nuclei. Paroxetine (50 nM) was used to block [125I]RTI-55 binding to 5-HT transporter sites. Initial experiments identified drugs that displaced [125I]RTI-55 binding with moderately low slope factors. Binding surface analysis of the interaction of 3 beta-(4-chlorophenyl)tropan-2 beta-carboxylic acid phenyl ester hydrochloride (RTI-113) and 3 beta-(4-iodophenyl)tropan-2 beta-carboxylic acid phenyl ester hydrochloride (RTI-122) with [125I]RTI-55 binding sites readily resolved two binding sites for [125I]RTI-55 with Kd values of 0.44 nM and 17 nM and Bmax values of 31 and 245 fmol/mg protein. Potent 5-HT and noradrenergic uptake inhibitors had low affinity for both sites. Whereas cocaine, CFT and WIN35,065-2 were 6.0-, 25- and 14-fold selective for the first site, benztropine, PCP and the novel pyrrole, (+-)-(2RS,3aSR,8bRS)-1,2,3,3a,4,8b-hexahydro- 2-benzyl-1-methylindeno-[1,2-b]pyrrole resorcylate [(+-)-HBMP, formerly called (+-)-RTI-4793-14], were moderately selective for the second site. A single binding site with the characteristics of site 1 was resolved using COS cells transiently expressing the cloned rat dopamine transporter. Lesion studies with 6-hydroxydopamine and 5,7-dihydroxytryptamine were conducted to test the hypothesis that site 1 and site 2 are physically distinct. The data showed that these neurotoxins differentially decreased [125I]RTI-55 binding to sites 1 and 2. The differential distribution of sites 1 and 2 in rat brain provides further support for this hypothesis. Viewed collectively, these data show that [125I]RTI-55 labels a novel binding site in rat brain membranes, termed DATsite2, which is not associated with the classic dopamine, serotonin or norepinephrine transporters.