The cardiac I(Ks) potassium channel is a macromolecular complex consisting of alpha-(KCNQ1) and beta-subunits (KCNE1) and the A kinase-anchoring protein (AKAP) Yotiao (AKAP-9), which recruits protein kinase A) and protein phosphatase 1 to the channel. Here, we have tested the hypothesis that specific cAMP phosphodiesterase (PDE) isoforms of the PDE4D family that are expressed in the heart are also part of the I(Ks) signaling complex and contribute to its regulation by cAMP. PDE4D isoforms co-immunoprecipitated with I(Ks) channels in hearts of mice expressing the I(Ks) channel. In myocytes isolated from these mice, I(Ks) was increased by pharmacological PDE inhibition. PDE4D3, but not PDE4D5, co-immunoprecipitated with the I(Ks) channel only in Chinese hamster ovary cells co-expressing AKAP-9, and PDE4D3, but not PDE4D5, co-immunoprecipitated with AKAP-9. Functional experiments in Chinese hamster ovary cells expressing AKAP-9 and either PDE4D3 or PDE4D5 isoforms revealed modulation of the I(Ks) response to cAMP by PDE4D3 but not PDE4D5. We conclude that PDE4D3, like protein kinase A and protein phosphatase 1, is recruited to the I(Ks) channel via AKAP-9 and contributes to its critical regulation by cAMP.