[125I]RTI-55 is a newly synthesized cocaine congener that may offer advantages over other ligands previously used to examine cocaine binding sites. However, the in vitro pharmacological and anatomical characterization of [125I]RTI-55 binding sites has not been previously performed in human brain. To determine the specificity, stability, and feasibility of [125I]RTI-55 for use in radioligand binding assays in postmortem human tissue, a series of experiments were performed characterizing [125I]RTI-55 binding sites in human brain using homogenized membrane preparations and quantitative autoradiography. Analysis of the association, dissociation, and saturation data favored two-phase processes. A curve-fitting analysis of the data derived in saturation experiments found a high-affinity site with KD = 66 +/- 35 pM and Bmax = 13.2 +/- 10.1 pmol/g of tissue and a low-affinity site with KD = 1.52 +/- 0.55 nM and Bmax of 47.5 +/- 11.2 pmol/g of tissue. Competition by ligands known to bind to the dopamine transporter showed a rank order of RTI-55 > GBR-12909 > maxindol > WIN 35428 > = methylphenidate > (-)-cocaine > buproprion > (+)-amphetamine. Binding to serotonergic sites was evaluated in the midbrain. Results of the saturation experiment performed autoradiographically in the midbrain showed a single site with KD = 370 +/- 84 pM. It appears that [125I]RTI-55 should be useful in further studies of the regulation of cocaine binding sites using postmortem human specimens.