AbstractKisspeptin receptor (KISS1R) signaling plays a critical role in the regulation of reproduction. We investigated the role of kisspeptin-stimulated KISS1R internalization, recycling, and degradation in the modulation of KISS1R signaling. Kisspeptin stimulation of Chinese hamster ovary or GT1–7 cells expressing KISS1R resulted in a biphasic increase in intracellular Ca2+ ([Ca2+]i), with a rapid acute increase followed by a more sustained second phase. In contrast, stimulation of the TRH receptor, another Gq/11-coupled receptor, resulted in a much smaller second-phase [Ca2+]i response. The KISS1R-mediated second-phase [Ca2+]i response was abolished by removal of kisspeptin from cell culture medium. Notably, the second-phase [Ca2+]i response was also inhibited by dynasore, brefeldin A, and phenylarsine oxide, which inhibit receptor internalization and recycling, suggesting that KISS1R trafficking contributes to the sustained [Ca2+]i response. We further demonstrated that KISS1R undergoes dynamic ligand-dependent and -independent recycling. We next investigated the fate of the internalized kisspeptin-KISS1R complex. Most internalized kisspeptin was released extracellularly in degraded form within 1 hour, suggesting rapid processing of the internalized kisspeptin-KISS1R complex. Using a biotinylation assay, we demonstrated that degradation of cell surface KISS1R was much slower than that of the internalized ligand, suggesting dissociated processing of the internalized kisspeptin-KISS1R complex. Taken together, our results suggest that the sustained calcium response to kisspeptin is dependent on the continued presence of extracellular ligand and is the result of dynamic KISS1R trafficking.