D 1 dopamine receptors are primary mediators of dopaminergic signaling in the CNS. These receptors internalize rapidly following agonist-induced activation, but the functional significance of this process is unknown. We investigated D 1 receptor endocytosis and signaling in HEK293 cells and cultured striatal neurons using real-time fluorescence imaging and cAMP biosensor technology. Agonist-induced activation of D 1 receptors promoted endocytosis of receptors with a time course overlapping that of acute cAMP accumulation. Inhibiting receptor endocytosis blunted acute D 1 receptor-mediated signaling in both dissociated cells and striatal slice preparations. Although endocytic inhibition markedly attenuated acute cAMP accumulation, inhibiting the subsequent recycling of receptors had no effect. Further, D 1 receptors localized in close proximity to endomembrane-associated trimeric G protein and adenylyl cyclase immediately after endocytosis. Together, these results suggest a previously unanticipated role of endocytosis, and the early endocytic pathway, in supporting rapid dopaminergic neurotransmission.