Neurotensin (NT) produces various stimulatory effects on dopaminergic neurons of the rat substantia nigra. To gain insight into the subcellular substrate for these effects, we compared by electron microscopy the distribution of immunoreactive high-affinity NT receptor proteins (NTRH) with that of high-affinity 125I-NT binding sites in this region of rat brain. Quantitative analysis showed a predominant association of immunogold and radioautographic labels with somata and dendrites of presumptive dopaminergic neurons, and a more modest localization in myelinated and unmyelinated axons and astrocytic leaflets. The distributions of immunoreactive NTRH and 125I-NT binding sites along somatodendritic plasma membranes were highly correlated and homogeneous, suggesting that membrane-targeted NTRH proteins were functional and predominantly extrasynaptic. Abundant immunocytochemically and radioautographically labeled receptors were also detected inside perikarya and dendrites. Within perikarya, these were found in comparable proportions over membranes of smooth endoplasmic reticulum and Golgi apparatus, suggesting that newly synthesized receptor proteins already possess the molecular and conformational properties required for effective ligand binding. By contrast, dendrites showed a proportionally higher concentration of immunolabeled than radiolabeled intracellular receptors. A fraction of these immunoreactive receptors were found in endosomes, suggesting that they had undergone ligand-induced internalization and were under a molecular conformation and/or in a physical location that precluded their recognition by and/or access to exogenous ligand. Our results provide the first evidence that electron microscopic immunocytochemistry of the NT receptor identifies sites for both the binding and trafficking of NT in the substantia nigra.