The cAMP-specific phosphodiesterase PDE4D5 can interact with the signalling scaffold proteins RACK (receptors for activated C-kinase) 1 and β-arrestin. Two-hybrid and co-immunoprecipitation analyses showed that RACK1 and β-arrestin interact with PDE4D5 in a mutually exclusive manner. Overlay studies with PDE4D5 scanning peptide array libraries showed that RACK1 and β-arrestin interact at overlapping sites within the unique N-terminal region of PDE4D5 and at distinct sites within the conserved PDE4 catalytic domain. Screening scanning alanine substitution peptide arrays, coupled with mutagenesis and truncation studies, allowed definition of RACK1 and β-arrestin interaction sites. Modelled on the PDE4D catalytic domain, these form distinct well-defined surface-exposed patches on helices-15–16, for RACK1, and helix-17 for β-arrestin. siRNA (small interfering RNA)-mediated knockdown of RACK1 in HEK-293 (human embryonic kidney) B2 cells increased β-arrestin-scaffolded PDE4D5 approx. 5-fold, increased PDE4D5 recruited to the β₂AR (β₂-adrenergic receptor) upon isoproterenol challenge approx. 4-fold and severely attenuated (approx. 4–5 fold) both isoproterenol-stimulated PKA (protein kinase A) phosphorylation of the β₂AR and activation of ERK (extracellular-signal-regulated kinase). The ability of a catalytically inactive form of PDE4D5 to exert a dominant negative effect in amplifying isoproterenol-stimulated ERK activation was ablated by a mutation that blocked the interaction of PDE4D5 with β-arrestin. In the present study, we show that the signalling scaffold proteins RACK1 and β-arrestin compete to sequester distinct ‘pools’ of PDE4D5. In this fashion, alterations in the level of RACK1 expression may act to modulate signal transduction mediated by the β₂AR.