TREK channels produce background currents that regulate cell excitability. These channels are sensitive to a wide variety of stimuli including polyunsaturated fatty acids (PUFAs), phospholipids, mechanical stretch, and intracellular acidification. They are inhibited by neurotransmitters, hormones, and pharmacological agents such as the antidepressant fluoxetine. TREK1 knockout mice have impaired PUFA-mediated neuroprotection to ischemia, reduced sensitivity to volatile anesthetics, altered perception of pain, and a depression-resistant phenotype. Here, we investigate TREK1 regulation by Gq-coupled receptors (GqPCR) and phospholipids. Several reports indicate that the C-terminal domain of TREK1 is a key regulatory domain. We developed a fluorescent-based technique that monitors the plasma membrane association of the C terminus of TREK1 in real time. Our fluorescence and functional experiments link the modulation of TREK1 channel function by internal pH, phospholipid, and GqPCRs to TREK1-C-terminal domain association to the plasma membrane, where increased association results in greater activity. In keeping with this relation, inhibition of TREK1 current by fluoxetine is found to be accompanied by dissociation of the C-terminal domain from the membrane.