The process of clathrin-coated vesicle (CCV) formation/disassembly involves numerous proteins that act cooperatively. Phosphorylation is an important regulatory mechanism governing protein interactions in CCVs, and many of the core and accessory proteins of the CCV machinery are reversibly phosphorylated in vivo. CK2 is highly enriched in CCVs and is capable of phosphorylating a number of peripheral membrane proteins involved in the process of clathrin-mediated endocytosis. At least some of these phosphorylation events have been shown to be inhibitory for CCV assembly, and CK2 has been shown to be inactive when associated with intact CCVs. Here we show that CCV membranes inhibit CK2 activity even after incubation in trypsin, indicating that a component of the lipid bilayer may be the inhibitory factor. Consistent with this, we showed that liposomes containing phosphatidylinositol phosphates inhibit the activity of CK2 and that CK2 binds to those liposomes. Notably, liposomes containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), a component of CCVs, bind CK2 and inhibit its activity. Furthermore, we showed that the binding of CK2 to PtdIns(4,5)P(2)-containing liposomes is via the active site of CK2, thus providing a molecular explanation for the inhibition of CK2 activity when it is bound to PtdIns(4,5)P(2)-containing liposomes. Thus CK2 is inactive in CCVs because of the fact that it is bound to the CCV membrane via an interaction between PtdIns(4,5)P(2) in the CCV membrane and the active site in CK2.