AbstractG protein-coupled receptors (GPCRs) transduce pleiotropic intracellular signals in a broad range of physiological responses and disease states. Activated GPCRs can undergo agonist-induced phosphorylation by G protein receptor kinases (GRKs) and second messenger-dependent protein kinases such as protein kinase A (PKA). Here, we characterize spatially segregated subpopulations of β₂-adrenergic receptor (β₂AR) undergoing selective phosphorylation by GRKs or PKA in a single cell. GRKs primarily label monomeric β₂ARs that undergo endocytosis, whereas PKA modifies dimeric β₂ARs that remain at the cell surface. In hippocampal neurons, PKA-phosphorylated β₂ARs are enriched in dendrites, whereas GRK-phosphorylated β₂ARs accumulate in soma, being excluded from dendrites in a neuron maturation-dependent manner. Moreover, we show that PKA-phosphorylated β₂ARs are necessary to augment the activity of L-type calcium channel. Collectively, these findings provide evidence that functionally distinct subpopulations of this prototypical GPCR exist in a single cell.