The cannabinoid 1 receptor (CB1R) is one of the most abundant seven transmembrane (7TM) spanning/G-protein-coupled receptors in the central nervous system and plays an important role in pain transmission, feeding, and the rewarding effects of cannabis. Tolerance to cannabinoids has been widely observed after long-term use, with concomitant receptor desensitization and/or down-regulation depending on the brain region studied. Several CB1R agonists promote receptor internalization after activation, but the postendocytic sorting of the receptor has not been studied in detail. Utilizing human embryonic kidney (HEK293) cells stably expressing the CB1R and primary cultured neurons expressing endogenous CB1R, we show that treatment with cannabinoid agonists results in CB1R degradation after endocytosis and that the G-protein-coupled receptor-associated sorting protein GASP1 plays a major role in the postendocytic sorting process. Thus these results may identify a molecular mechanism underlying tolerance and receptor down-regulation after long-term use of cannabinoids.--Martini, L., Waldhoer, M., Pusch, M., Kharazia, V., Fong, J., Lee, J. H., Freissmuth, C., Whistler, J. L. Ligand-induced down-regulation of the cannabinoid 1 receptor is mediated by the G-protein-coupled receptor-associated sorting protein GASP1.