Membrane-bound anionic phospholipids such as phosphatidylinositols have the capacity to modulate ATP-sensitive potassium (K_ATP) channels through a mechanism involving long-range electrostatic interaction between the lipid headgroup and channel. However, it has not yet been determined whether the multiple effects of phosphatidylinositols reported in the literature all result from this general electrostatic interaction or require a specific headgroup structure. The present study investigated whether phosphatidic acid (PA), an anionic phospholipid substantially different in structure from phosphatidylinositols, evokes effects similar to phosphatidylinositols on native K_ATP channels of rat heart and heterogeneous Kir6.2/SUR2A channels. Channels treated with PA (0.2-1 mg/ml applied to the cytoplasmic side of the membrane) exhibited higher activity, lower sensitivity to ATP inhibition, less Mg²⁺-dependent nucleotide stimulation, and poor sulfonylurea inhibition. These effects match the spectrum of phosphatidylinositols' effects, but, in addition, PA also induced a novel pattern in gating kinetics, represented by a decreased mean open time (from 12.2 ± 2.0 to 3.3 ± 0.7 ms). This impact on gating kinetics clearly distinguishes PA's effects from those of phosphatidylinositols. Results indicate that multiple effects of anionic phospholipids on K_ATP channels are related phenomena and can likely be attributed to a common mechanism, but additional specific effects due to other mechanisms may also coincide.