Inward rectifier K⁺ channels, which modulate electrical activity in many cell types, are regulated by protein kinases,, guanine-nucleotide-binding proteins (G proteins)^,,, and probably actin cytoskeleton. Generation of phosphatidylinositol 4,5-bisphosphate (PIP₂) by ATP-dependent lipid kinases is known to activate inward rectifier K⁺ channels in cardiac membrane patches. Herewe report that several cloned inward rectifier K⁺ channels directly bind PIP₂, and that this binding correlates with channel activity. Application of ATP or PIP₂ liposomes activates the cloned channels. Stabilized by lipid phosphatase inhibitors, PIP₂ antibodies potently inhibit each channel with a unique rate (GIRK1/4 (-) ≈ GIRK2 ≫ IRK1 ≈ ROMK ). Consistent with the faster dissociation of PIP₂ from the GIRK channels, the carboxy terminus of GIRK1 binds ³H-PIP₂ liposomes more weakly than does that of IRK1 or ROMK1. Mutation of a conserved arginine to glutamine at position 188 reduces the ability of ROMK1 to bind PIP₂ and increases its sensitivity to inhibition by PIP₂ antibodies. Interactions between GIRK channels and PIP₂ are modulated by the βγ subunits of the G protein (Gβγ). When GIRK1/4 channels are allowed to run down completely, they are not activated by addition of Gβγ alone, but application of PIP₂ activates them in minutes without Gβγ and in just seconds with Gβγ. Finally, coexpression of Gβγ with GIRK channels slows the inhibition of K⁺ currents by PIP₂ antibodies by more than 10-fold. Thus Gβγ activates GIRK channels by stabilizing interactions between PIP₂ and the K⁺ channel.