The present studies examined the characteristics of fetal (gestational day 20) and adult rat brain cocaine recognition sites labeled with the potent cocaine congener [125I]RTI-55 [3 beta-(4-iodophenyl)-tropane-2 beta-carboxylic acid methyl ester]. Saturation analyses of [125I]RTI-55 binding to membrane fractions from both fetal and adult whole-brain yielded curvilinear Scatchard plots that were resolved by nonlinear curve-fitting into high- and low-affinity components. Mean Kd values were 0.13 nM and 12 nM for fetal brain high- and low-affinity sites, respectively, compared to 0.26 and 18 nM for adult brain. The Kd for high-affinity binding was significantly different between the groups, suggesting a possible developmental change in the properties of [125I]RTI-55 binding sites. Drug displacement studies with various monoamine uptake inhibitors indicated that at a 10 pM concentration of [125I]RTI-55, almost all binding to fetal brain membranes could be accounted for by interaction with the serotonin (5-HT) and (to a lesser extent) dopamine (DA) transporters. This conclusion was supported by autoradiographic studies of both adult and fetal brain, demonstrating a predominance of [125I]RTI-55 binding in areas with high densities of 5-HT and/or DA uptake sites. The present results are in accordance with our previous demonstration of [3H]cocaine binding sites in fetal brain (Meyer et al., 1993) and further suggest that [125I]RTI-55 should be a useful ligand for assessing the effects of prenatal cocaine exposure on the subsequent development of cocaine recognition sites on the DA and 5-HT transporters.