Recent work has established membrane phospholipids such as phosphatidylinositol-4,5-bisphosphate (PIP(2)) as potent regulators of K(ATP) channels controlling open probability and ATP sensitivity. We here investigated the effects of phospholipids on the pharmacological properties of cardiac type K(ATP) (Kir6.2/SUR2A) channels. In excised membrane patches K(ATP) channels showed considerable variability in sensitivity to glibenclamide and ATP. Application of the phosphatidylinositol phosphates (PIPs) phosphatidylinositiol-4-phosphate, PIP(2), and phosphatidylinositol-3,4,5-trisphosphate reduced sensitivity to ATP and glibenclamide closely resembling the native variability. Insertion of the patch back into the oocyte (patch-cramming) restored high ATP and glibenclamide sensitivity, indicating reversible modulation of K(ATP) channels via endogenous PIPs-degrading enzymes. Thus, the observed variability seemed to result from differences in the membrane phospholipid content. PIP(2) also diminished activation of K(ATP) channels by the K(+) channel openers (KCOs) cromakalim and P1075. The properties mediated by the sulphonylurea receptor (sensitivity to sulfonylureas and KCOs) seemed to be modulated by PIPs via a different mechanism than ATP inhibition mediated by the Kir6.2 subunits. First, polycations abolished the effect of PIP(2) on ATP inhibition consistent with an electrostatic mechanism but only weakly affected glibenclamide inhibition and activation by KCOs. Second, PIP(2) had clearly distinct effects on the concentration-response curves for ATP and glibenclamide. However, PIPs seemed to mediate the different effects via the Kir6.2 subunits because a mutation in Kir6.2 (R176A) attenuated simultaneously the effects of PIP(2) on ATP and glibenclamide inhibition. Finally, experiments with various lipids revealed structural features necessary to modulate K(ATP) channel properties and an artificial lipid (dioleoylglycerol-succinyl-nitriloacetic acid) that mimicked the effects of PIPs on K(ATP) channels.