Expression of the tandem P domain K⁺ channel, hTREK1, is limited almost exclusively to the central nervous system where ambient pO² can be as low as 20 mmHg. We have previously shown that this level of hypoxia evokes a maximal inhibitory influence on recombinant hTREK1 and occludes the activation by arachidonic acid; this has cast doubt on the idea that TREK1 activation during brain ischemia could facilitate neuroprotection via hyperpolarising neurons in which it is expressed. Using both whole cell and cell-attached patch clamp configurations, we now show that the action of another potent TREK activator and ischemia-related event, intracellular acidification, is similarly without effect during compromised O² availability. This occlusion is observed in either recording condition, and even the concerted actions of both arachidonic acid and intracellular acidosis are unable to activate hTREK1 during hypoxia. Conversely, intracellular alkalinization is a potent channel inhibitor and hypoxia does not reverse this inhibition. However, increases in intracellular pH are unable to occlude either arachidonic acid activation or hypoxic inhibition. These data highlight two important points. Firstly, during hypoxia, modulation of hTREK1 cannot be accomplished by parameters known to be perturbed in brain ischemia (increased extracellular fatty acids and intracellular acidification). Secondly, the mechanism of regulation by intracellular alkalinization is distinct from the overlapping structural requirements known to exist for regulation by arachidonic acid, membrane distortion and acidosis. Thus, it seems likely that hTREK1 regulation in the brain will be physiologically more relevant during alkalosis than during ischemia or acidosis.