β-Arrestin proteins play a dual role in regulating G protein-coupled receptor (GPCR) responsiveness by contributing to both receptor desensitization and internalization. Recently, β-arrestins were also shown to be critical determinants for β 2 -adrenergic receptor (β 2 AR) resensitization. This was demonstrated by overexpressing wild-type β-arrestins to rescue the resensitization-defect of a β 2 AR (Y326A) mutant (gain of function) and overexpressing a dominant-negative β-arrestin inhibitor of β 2 AR sequestration to impair β 2 AR dephosphorylation and resensitization (loss of function). Moreover, the ability of the β 2 AR to resensitize in different cell types was shown to be dependent upon β-arrestin expression levels. To further study the mechanisms underlying β-arrestin function, green fluorescent protein was coupled to β-arrestin2 (βarr 2 GFP), thus allowing the real-time visualization of the agonist-dependent trafficking of β-arrestin in living cells. βarr 2 GFP translocation from the cytoplasm to the plasma membrane proceeded with a time course, sensitivity and specificity that was indistinguishable from the most sensitive second messenger readout systems. βarr 2 GFP translocation was GRK-dependent and was demonstrated for 16 different ligand-activated GPCRs. Because β-arrestin binding is a common divergent step in GPCR signalling, this assay represents a universal methodology for screening orphan receptors, GRK inhibitors and novel GPCR ligands. Moreover, βarr 2 GFP provides a valuable new tool to dissect the biological function and regulation of β-arrestin proteins.