This paper compares the localization of neurotensin receptors and of endopeptidase 24–16, a peptidase likely involved in the inactivation of neurotensin in primary cultures of neurons. Neurotensin binding sites were radiolabeled with 125I-Tyr3-neurotensin, whereas endopeptidase 24– 16 was stained by immunohistochemical techniques using a monospecific polyclonal antibody. Endopeptidase 24–16 is present in 80–85% of the nondifferentiated neurons. The proportion of immunoreactive neurons decreased during maturation to reach 35–40% after 4–8 d of culture. By contrast, neurotensin receptors were not detectable in nondifferentiated cells and appear during maturation. Specific 125I- Tyr3-neurotensin labeling is maximal after 4 d of culture and is located on about 10% of differentiated neurons. Double-labeling experiments show that about 90% of cortical, hypothalamic, and mesencephalic neurons bearing the neurotensin receptor also contained endopeptidase 24–16, supporting the hypothesis that one of the functions of endopeptidase 24–16 is the physiological inactivation of neurotensin. However, the presence of endopeptidase 24–16 on numerous neurons that do not contain neurotensin receptors also suggests that the enzyme could be involved in the degradation and/or maturation of other neuropeptides.