TREK-1 is a member of the K_2P channel family that is mechano-, heat-, pH-,anaesthetic- and lipid-sensitive. It is highly expressed in the central nervous system and probably encodes one of the arachidonic acid activated K⁺-channels. Polyunsaturated fatty acids and lysophospholipids protect the brain against ischemia. Since both are openers of TREK-1, it has been suggested that this K_2P channel is directly involved in neuroprotection (Patel & Honoré, 2001). Recently, however this view has been challenged by a report claiming that TREK-1 and its activation by arachidonic acid is inhibited by hypoxia (Miller et al. 2003). We have reinvestigated this phenomenon. HEK 293 cells were transiently transfected with plasmids containing DNA encoding mouse TREK-1 ( pCi IRES-CD8-mTREK-1) and enhanced green fluorescent protein (pCi IRES-EGFP-mTREK-1). Whole cell voltage-clamp recordings were conducted in HEPES buffered media at room temperature 48-72 hrs post transfection. Pipette filling solutions contained 5 mM ATP. In preliminary experiments the effects of arachidonic acid upon TREK-1 appeared to be greatly suppressed by bubbling solutions with a wide range of gas mixtures including air. Upon further investigation we found that gas bubbling resulted in the loss of 78% ± 1% (mean ± SEM, n=4) of 3H arachidonic acid from the bulk phase of solution within an hour. We suggest that this may be due to the amphipathic nature of arachidonic acid favouring its redistribution to the air water interface aided by the passage of bubbles through solution. By using a gas equilibration method which avoided bubbling induced loss of arachidonic acid from solution, we found mTREK-1 to be strongly activated by arachidonic acid under both normoxic and hypoxic conditions. Mean mTREK-1 current measured at 0 mV in the presence of 10 μM arachidonic acid was 4.6 ± 1.2 nA in air equilibrated solutions and 4.7 ± 1.0 nA in hypoxic solutions (N₂ equilibrated, PO₂ < 4 torr); both values were significantly (P < 0.02, paired Student's t test) greater than their respective control levels (0.37 ± 0.1 nA in air & 0.4 ± 0.1 nA in N₂, n=6). Indeed hypoxia had no significant effect upon mTREK-1 currents under either control conditions or in the presence of arachidonic acid. These data demonstrate that TREK-1 is strongly activated by arachidonic acid even under hypoxic conditions and thus support the proposed role for TREK-1 in ischemic neuroprotection.