Many ion channels are modulated by phosphatidylinositol 4,5-bisphosphate (PIP2), but studies examining the PIP2 dependence of channel activity have been limited to cell expression systems, which present difficulties for controlling membrane composition. We have characterized the PIP2 dependence of purified human Kir2.1 and Kir2.2 activity using 86Rb+ flux and patch clamp assays in liposomes of defined composition. We definitively show that these channels are directly activated by PIP2 and that PIP2 is absolutely required in the membrane for channel activity. The results provide the first quantitative description of the dependence of eukaryotic Kir channel function on PIP2 levels in the membrane; Kir2.1 shows measureable activity in as little as 0.01% PIP 2, and open probability increases to ∼0.4 at 1% PIP2. Activation of Kir2.1 by phosphatidylinositol phosphates is also highly selective for PIP2; PI, PI(4)P, and PI(5)P do not activate channels, and PI(3,4,5)P3 causes minimal activity. The PIP2 dependence of eukaryotic Kir activity is almost exactly opposite that of KirBac1.1, which shows marked inhibition by PIP2. This raises the interesting hypothesis that PIP2 activation of eukaryotic channels reflects an evolutionary adaptation of the channel to the appearance of PIP2 in the eukaryotic cell membrane.