The regulation of neurotensin receptors was studied in vitro in primary cultures of neuronal cells. High affinity receptors for [³H]neurotensin were found in homogenates and at the cell surface of intact neurons cultured from the brain of rat embryos. When intact cells were incubated with 3 nM neurotensin (1–13), a rapid decrease in [³H]neurotensin binding was observed; about 60% of neurotensin receptors disappeared from the cell surface in less than 15 min. This corresponded to a reduction of the B_max value without a change in the binding affinity. The decrease in neurotensin receptor number was also induced by the active fragment (8–13) of neurotensin but not by its inactive fragment (1–8). It was partially inhibited by bacitracin, at concentrations which are known to interact with receptor internalization, and was not detected when intact cells were incubated at 0–4° with the unlabeled peptide. When intact neurons were incubated with [³H]neurotensin, there was a rapid ligand uptake and the kinetics of endocytosis were similar to those of the cell surface receptor disappearance. Once endocytosed, [³H]neurotensin could not be released (or displaced) from either intact neurons or homogenates, suggesting the sequestration of the labeled peptide in vesicles or other subcellular structures. Therefore, the present results suggest that the rapid agonist-induced decrease in the number of neurotensin receptors from the cell surface corresponds to an internalization process which involves a simultaneous receptormediated peptide endocytosis.