β-Arrestins bind to activated G protein-coupled receptor kinase-phosphorylated receptors, which leads to their desensitization with respect to G proteins, internalization via clathrin-coated pits, and signaling via a growing list of “scaffolded” pathways. To facilitate the discovery of novel adaptor and signaling roles of β-arrestins, we have developed and validated a generally applicable interfering RNA approach for selectively suppressing β-arrestins 1 or 2 expression by up to 95%. β-Arrestin depletion in HEK293 cells leads to enhanced cAMP generation in response to β₂-adrenergic receptor stimulation, markedly reduced β₂-adrenergic receptor and angiotensin II receptor internalization and impaired activation of the MAP kinases ERK 1 and 2 by angiotensin II. This approach should allow discovery of novel signaling and regulatory roles for the β-arrestins in many seven-membrane-spanning receptor systems.