Mazindol is a potent inhibitor of neuronal dopamine (DA) and norepinephrine (NE) uptake. DA and NE uptake sites in rat brain have been differentially visualized using [3H]mazindol autoradiography. At appropriate concentrations, desipramine (DMI) selectively inhibits [3H]mazindol binding to NE uptake sites without significantly affecting binding to DA uptake sites. The localization of DMI-insensitive specific [3H] mazindol binding, reflecting DA uptake sites, is densest in the caudate-putamen, the nucleus accumbens, the olfactory tubercle, the subthalamic nucleus, the ventral tegmental area, the substantia nigra (SN) pars compacta, and the anterior olfactory nuclei. In contrast, the localization of DMI-sensitive specific [3H]mazindol binding, representing NE uptake sites, is densest in the locus coeruleus, the nucleus of the solitary tract, the bed nucleus of the stria terminalis, the paraventricular and periventricular nuclei of the hypothalamus, and the anteroventral thalamus. The distribution of DMI-insensitive specific [3H]mazindol binding closely parallels that of dopaminergic terminal and somatodendritic regions, while the distribution of DMI-sensitive specific [3H]mazindol binding correlates well with the regional localization of noradrenergic terminals and cell bodies. Injection of 6-hydroxydopamine, ibotenic acid, or colchicine into the SN decreases [3H]mazindol binding to DA uptake sites in the ipsilateral caudate-putamen by 85%. In contrast, ibotenic acid lesions of the caudate-putamen do not reduce [3H]mazindol binding to either the ipsilateral or contralateral caudate-putamen. Thus, the DA uptake sites in the caudate-putamen are located on the presynaptic terminals of dopaminergic axons originating from the SN.