Neurotensin is present in high quantity in the hypothalamus, where it regulates pituitary hormone secretion. A relationship between dopaminergic and neurotensinergic systems has been suggested in the hypothalamus in studies showing an effect of neurotensin on tuberoinfundibular dopaminergic neurons. In order to determine the anatomical basis of such interactions, primary cultures of rat hypothalamic neurons were used. Tyrosine hydroxylase and neurotensin containing cells were identified by immunocytochemistry and neurotensin binding sites by [125I]Tyr3-neurotensin autoradiography. Colocalization studies showed that neurotensin immunoreactivity was present in 16% of tyrosine hydroxylase-positive cells, and that these neurotensinhyrosine hydroxylase neurons represented more than half (58%) of the neurotensinergic population. Five percent of the tyrosine hydroxylase-positive cells had neurotensin binding sites, suggesting that only a restricted number of hypothalamic dopaminergic neurons is responsive to neurotensin. Neurotensin binding sites were also found on some neurotensin-positive cells, demonstrating for the first time the presence of autoreceptors for this peptide on neurons. These results in primary cultures provide a cellular basis for direct effects of neurotensin on a subpopulation of hypothalamic dopaminergic cells, and support the possibility of an autocrine action of neurotensin in the hypothalamus.