One of the defining properties of beta2-adrenergic receptor (beta(2)AR) signaling is the transient and rapidly reversed accumulation of cAMP. Here we have investigated the contribution of different PDE4 proteins to the generation of this transient response. To this aim, mouse embryonic fibroblasts deficient in PDE4A, PDE4B, or PDE4D were generated, and the regulation of PDE activity, the accumulation of cAMP, and CREB phosphorylation in response to isoproterenol were monitored. Ablation of PDE4D, but not PDE4A or PDE4B, had a major effect on the beta-agonist-induced PDE activation, with only a minimal increase in PDE activity being retained in PDE4D knock-out (KO) cells. Accumulation of cAMP was markedly enhanced, and the kinetics of cAMP accumulation were altered in their properties in PDE4DKO but not PDE4BKO cells. Modest effects were observed in PDE4AKO mouse embryonic fibroblasts. The return to basal levels of both cAMP accumulation and CREB phosphorylation was greatly delayed in the PDE4DKO cells, suggesting that PDE4D is critical for dissipation of the beta2AR stimulus. This effect of PDE4D ablation was in large part due to inactivation of a negative feedback mechanism consisting of the PKA-mediated activation of PDE4D in response to elevated cAMP levels, as indicated by experiments using the cAMP-dependent protein kinase inhibitors H89 and PKI. Finally, PDE4D ablation affected the kinetics of beta2AR desensitization as well as the interaction of the receptor with Galphai. These findings demonstrate that PDE4D plays a major role in shaping the beta2AR signal.