Molecular mechanisms regulating the development of physiological and behavioral tolerance to cannabinoids are not well understood. Two cellular correlates implicated in the development and maintenance of tolerance are CB₁ cannabinoid receptor internalization and uncoupling of receptor signal transduction. Both processes have been proposed as mediators of tolerance because of observations that chronic Δ⁹-tetrahydrocannabinol (THC) treatment causes both region-specific decreases in CB₁ receptors and G-protein coupling in the brain. To determine the balance of these two processes in regulating CB₁ receptor signaling during sustained receptor stimulation, we evaluated the parameters affecting ERK1/2 MAP kinase activity in HEK293 cells stably expressing CB₁ receptors. CB₁ receptor stimulation by the potent CB₁ receptor agonist, CP 55,940 transiently activated ERK1/2. To determine if CB₁ receptor desensitization or internalization was responsible for the transient nature of ERK1/2 activation, we evaluated ERK1/2 phosphorylation in HEK293 cells expressing a desensitization-deficient CB₁ receptor (S426A/S430A CB₁). Here, the duration of S426A/S430A CB₁ receptor-mediated activation of ERK1/2 was markedly prolonged relative to wild-type receptors, and was dynamically reversed by SR141716A. Interestingly, the S426A/S430A CB₁ receptor was still able to recruit βarrestin-2, a key mediator of receptor desensitization, to the cell surface following agonist activation. In contrast to a central role for desensitization, pharmacological and genetic approaches suggested CB₁ receptor internalization is dispensable in the transient activation of ERK1/2. This study indicates that the duration of ERK1/2 activation by CB₁ receptors is regulated by receptor desensitization and underscores the importance of G-protein uncoupling in the regulation of CB₁ receptor signaling.