When rat cultured neurons were incubated with unlabelled neurotensin (3 nM) for 1 or 24 h at 37 degrees C, the [3H]-neurotensin specific binding measured in cell homogenates was decreased to about 35 and 65% of control values, respectively. In these experiments, the decreases in binding corresponded to reductions of Bmax values without changes in the affinity. The slow neurotensin-induced receptor down-regulation is thought to result from receptor degradation since it was reduced by the lysosomotropic drugs chloroquine and methylamine and because no change in neurotensin mRNA level could be measured after the neurotensin stimulation. After their internalization, receptors slowly reappeared at the cell surface after further incubation in the absence of the peptide. Such receptor reappearance was prevented in the presence of the protein synthesis inhibitor cycloheximide and is therefore thought to result from new synthesis and not from recycling of internalized receptors. These results indicate that the neurotensin-induced receptor internalization in cultured neurons is irreversible and that it is followed by a down-regulation of the receptor through a degradative process.