ROMK inward-rectifier K⁺ channels control renal K⁺ secretion. The activity of ROMK is regulated by protein kinase A (PKA), but the molecular mechanism for regulation is unknown. Having found that direct interaction with membrane phosphatidylinositol 4,5-bisphosphate (PIP₂) is essential for channel activation, we investigate here the role of PIP₂ in regulation of ROMK1 by PKA. By using adenosine-5′-[γ-thio]triphosphate) (ATP[γS]) as the substrate, we found that PKA does not directly activate ROMK1 channels in membranes that are devoid of PIP₂. Rather, phosphorylation by PKA + ATP[γS] lowers the concentration of PIP₂ necessary for activation of the channels. In solution-binding assays, anti-PIP₂ antibodies bind PIP₂ and prevent PIP₂–channel interaction. In inside-out membrane patches, antibodies inhibit the activity of the channels. PKA treatment then decreases the sensitivity of ROMK1 for inhibition by the antibodies, indicating an enhanced interaction between PIP₂ and the phosphorylated channels. Conversely, mutation of the PKA phosphorylation sites in ROMK1 decreases PIP₂ interaction with the channels. Thus, PKA activates ROMK1 channels by enhancing PIP₂–channel interaction.