1 Neuropeptide Y Y(1) receptors are known to internalize following the binding of agonists. In the present study, a pseudopeptide Y(1) receptor antagonist, homodimeric Ile-Glu-Pro-Dpr-Tyr-Arg-Leu-Arg-Tyr-CONH(2) (GR231118), also induced Y(1) receptor internalization in human embryonic kidney (HEK293) cells. 2 We demonstrated first that both specifically bound radiolabeled antagonist ([(125)I]GR231118) and agonist ([(125)I][Leu(31), Pro(34)]PYY) underwent receptor-mediated sequestration/internalization in transfected HEK293 cells. 3 Agonist-induced Y(1) receptor internalization was dependent on clathrin-coated pits and was regulated in part by Gi/o-protein activation as revealed by pertussin toxin sensitivity. In contrast, antagonist-induced sequestration of Y(1) receptors was partly dependent on clathrin-coated pits, but independent from Gi/o-protein activation. 4 Exposure to high concentrations of agonist or antagonist caused a 50 and 75% loss of cell surface binding, respectively. The loss caused by the agonist rapidly recovered. This phenomenon was blocked by monensin, an inhibitor of endosome acidification, suggesting that cell surface receptor recovery is due to recycling. In contrast to the agonist, GR231118 induced a long-lasting sequestration of Y(1) receptors in HEK293 cells. 5 Immunofluorescence labeling indicated that following 40 min of incubation with either the agonist or the antagonist, Y(1) receptors followed markedly different intercellular trafficking pathways. 6 Taken together, these findings provided evidence that a pseudopeptide Y(1) receptor antagonist can induce long-lasting disappearance of cell surface receptors through a pathway distinct from the classical endocytic/recycling pathway followed by stimulation with an agonist.