TREK1, two pore domain potassium (K2P) channels are responsible for background currents that regulate neuronal excitability. Deletion of TREK1 has previously been shown to result in a depression-resistant phenotype in mice (Heurteaux et al. 2006). In this study we investigated the effect of the tricyclic antidepressant, amitriptyline, on TREK1 channels. Additionally, amitriptyline is used for relief of neuropathic pain and TREK-1 is also involved in pain perception (Alloui et al. 2006). tsA-201 cells were transiently transfected with wild type and mutated K2P channels. The whole cell patch clamp technique was used to obtain current recordings. Homology models for TREK1 were constructed using Modeller. Amitriptyline was docked to these models using the MOE program. Amitriptyline (50 μM) significantly inhibited WT TREK1 channel current by 77.4 ± 3.7 % (n=9). While the inhibitory effect of amitriptyline was reversible, on wash, it was often followed by an over-recovery of current to above the baseline level. We have previously investigated the effect of the antidepressant, citalopram, on TREK1, and found that it inhibited the channel. Subsequently we identified a number of amino acids in both the pore region and transmembrane domains M2 and M4 of TREK1 that were important for its effect (Al-Moubarak et al. 2013). Interestingly, mutations of several amino acids, T142A, L289A and T251A, which reduced the effectiveness of citalopram had no effect on the observed inhibition by amitriptyline. Other mutations, L169A and I167A had no effect on inhibition by either compound. Mutation L174A (in the M2 transmembrane domain of TREK1), however, significantly reduced inhibition by both citalopram (Al-Moubarak et al 2013) and amitriptyline (50 μM, 49.0 ± 3.1 %, n=6, p < 0.05). As previously found for citalopram, the E306A gain of function mutation completely prevented inhibition by amitriptyline. Docking of amitriptyline into the TREK1 homology model, guided by the obtained electrophysiology results, predicts a different binding profile for amitriptyline compared to citalopram. Thus we have shown that amitriptyline is a potent inhibitor of TREK1 potassium channels. This action may contribute to the therapeutic usefulness of this compound in both the treatment of depression and pain.