Desensitization and internalization of G protein-coupled receptors observed after agonist activation are considered two important regulatory processes of receptor transduction. Endogenous human delta-opioid receptors (hDOR) are differentially regulated in terms of desensitization by peptide ([d-Pen2,5]enkephalin (DPDPE) and Deltorphin I) and alkaloid (etorphine) agonists in the neuroblastoma cell line SK-N-BE (Allouche, S., Roussel, M., Marie, N., and Jauzac, P. (1999) Eur. J. Pharmacol. 371, 235-240). In the present study, we examined the role of hDOR internalization and down-regulation in this differential desensitization. Sustained activation by peptides for 30 min caused a marked decrease of both [3H]diprenorphine binding sites and hDOR immunoreactivity, observed in a Western blot, whereas a moderate reduction by 30% was observed after a 30- and 60-min etorphine exposure in binding experiments without opioid receptor degradation. Using fluorescence microscopy, we visualized hDOR internalization promoted by different agonists in SK-N-BE cells expressing FLAG-tagged hDOR. Agonist withdrawal results in a greater recycling process correlated with a stronger hDOR resensitization after etorphine treatment compared with DPDPE or Deltorphin I, as shown in binding, immunocytochemical, and functional experiments. This suggests a distinct sorting of opioid receptors after their internalization. We demonstrated a lysosomal hDOR targeting upon peptides by using chloroquine in binding, Western blot, and immunocytochemical experiments and by colocalization of this receptor with a late endosome marker. In contrast, when the recycling endosome blocker monensin was used, acceleration of desensitization associated with a strong intracellular immunostaining was observed upon etorphine treatment. The possibility of separate endocytic pathways responsible for the differential sorting of hDOR upon peptide and alkaloid ligand exposure was ruled out by binding and immunocytochemical experiments using sucrose hypertonic solution. First, these results showed complex relationships between hDOR internalization/down-regulation and desensitization. Second, we demonstrated for the first time that the same receptor could undergo a distinct sorting after internalization by peptide and alkaloid agonists.