In this study, a sequential analysis of pathways involved in the regulation of GH secretagogue receptor subtype 1a (GHSR-1a) signaling has been undertaken to characterize the process of rapid desensitization that is observed after ghrelin binding. This process was evaluated by studying the binding of [(125)I]ghrelin, measurement of intracellular calcium mobilization, and confocal microscopy. The results indicate that GHSR-1a is mainly localized at the plasma membrane under unstimulated conditions and rapidly desensitizes after stimulation. The agonist-dependent desensitization is not mediated by protein kinase C because phorbol ester, phorbol-12-myristate-13-acetate, failed to block the ghrelin-induced calcium response. The ghrelin/GHSR-1a complex progressively disappears from the plasma membrane after 20 min exposure to ghrelin and accumulates in the perinuclear region after 60 min. Colocalization of the internalized GHSR-1a with the early endosome marker (EEA1) after 20 min exposure to ghrelin suggests that endocytosis occurs via clathrin-coated pits, which is consistent with the lack of internalization of this receptor observed after potassium depletion. Different from other G protein-coupled receptors, GHSR-1a showed slow recycling. Surface binding slowly recovered after agonist treatment and returned to control levels within 360 min. Furthermore, inhibition of vacuolar H(+)-ATPases prevented recycling of the receptor, suggesting that the nondissociation of the ligand/receptor complex is responsible for this effect. The GHSR-1a internalization may explain the characteristic physiological responses mediated by this receptor.