TREK-1 is an unconventional K(+) channel that is activated by both physical and chemical stimuli. In this study, we show that the inner leaflet membrane phospholipids, including PIP(2), exert a mixed stimulatory and inhibitory effect on TREK-1. Intra-cellular phospholipids inhibit basal channel activity and activation by membrane stretch, intra-cellular acidosis and arachidonic acid. However, binding of endogenous negative inner leaflet phospholipids with poly-lysine reduces inhibition and reveals channel stimulation by exogenous intra-cellular phospholipids. A similar effect is observed with PI, PE, PS and PA, unlike DG, demonstrating that the phosphate at position 3 is required although the global charge of the molecule is not critical. Inhibition depends on the distal C-terminal domain that conditions channel mechano-sensitivity, but is independent of the positively charged PIP(2) stimulatory site in the proximal C-terminal domain. This is, to our knowledge, the first report of an ion channel dually regulated by membrane phospholipids.