BACKGROUND AND PURPOSE TREK-1 is a mechanosensitive channel also activated by polyunsaturated fatty acids (PUFAs). TREK-1 activation by PUFAs is supposed to be linked to changes in membrane tension following PUFAs insertion. Their ability to insert into the membrane increases with their hydrophobicity and thus the length of the carbon chain. EXPERIMENTAL APPROACH In this study, we compared the effect of multiple fatty acids and ML402 in both whole-cell and inside-out configurations of the patch-clamp technique. KEY RESULTS First, we showed a variable TREK-1 activation by PUFAs related to the variable constitutive activity of TREK-1. Then, we observed no correlation between TREK-1 activation and acyl chain length or number of double bonds suggesting that the bilayer-couple hypothesis cannot explain by itself the activation of TREK-1 by PUFAs. The membrane fluidity measurement is not modified by PUFAs at 10 µM. The spectral shift analysis in TREK-1-enriched microsomes indicates a KD,TREK1 at 44 µM of C22:6 n-3. PUFAs display the same activation and reversible kinetics than the direct activator ML402 and activate TREK-1 in both whole-cell and inside-out configurations of patch-clamp suggesting that the binding site of PUFAs is accessible from both sides of the membrane, as for ML402. CONCLUSION AND IMPLICATIONS Finally, we proposed a two steps mechanism for TREK-1 activation by PUFAs: first, insertion into the membrane, with no fluidity or curvature modifications at 10 µM, and then interaction with TREK-1 channel to open it.