Several lines of anatomical, biochemical, and pharmacological evidence have suggested that specific high affinity neurotensin binding sites are associated with dopamine-containing neurons in the rat ventral tegmentum. In the present study we confirmed and quantified the extent of this association by combining monoiodinated neurotensin radioautography and tyrosine hydroxylase immunohistochemistry on adjacent 5-10 microns-thick midbrain sections. We found that 95-100% tyrosine hydroxylase-immunoreactive neurons detected in all subdivisions of the substantia nigra (pars compacta, pars lateralis, and pars reticulata) exhibited intense 125I-neurotensin labeling in adjacent light microscopic radioautographs. Tyrosine hydroxylase-positive dendrites radiating downward from compacta neurons were also heavily labeled throughout the pars reticulata. In the paranigral subdivision of the ventral tegmental area, silver grains were evenly distributed over neuropil and perikarya and therefore could not be readily attributed to any given tyrosine hydroxylase-positive element. In contrast, within the parabrachial pigmentous subdivision of the ventral tegmental area, 80-90% of the tyrosine hydroxylase-immunoreactive somata and proximal processes were clearly in register with 125I-neurotensin labeled cells. Finally, all tightly packed TH-positive neurons in the interfascicular nucleus showed intense 125I-neurotensin labeling. The vast majority of the neurotensin binding sites observed in the ventral midbrain tegmentum were of the high affinity, physiologically active receptor type since levocabastine, a selective blocker of the low affinity neurotensin binding component, had minimal effect on the binding density in any of the midbrain regions examined. The present results demonstrate an extensive overlap between specific, high affinity neurotensin binding sites and dopamine perikarya and dendrites in the rat ventral tegmentum, and thereby provide a direct anatomical substrate for observed neurotensin-dopamine interactions in the mesocorticolimbic and nigrostriatal projection systems.