GABA_A receptors are the major inhibitory neurotransmitter receptors. The endocannabinoid system is a lipid signaling network that modulates different brain functions. A direct molecular interaction between the two systems is documented. The endocannabinoid 2-arachidonoyl glycerol (2- AG) potentiates GABA_A receptors at low concentrations of GABA, thus identifying 2-AG as an endogenous ligand. 2-AG acts super-additive with the neurosteroid THDOC (3a, 21-dihydroxy-5a- pregnan-20-one) and modulates d subunit containing receptors, known to be located extrasynaptically and to respond to neurosteroids. 2-AG inhibits motility in CB1/CB2 cannabinoid receptor double knockout mice, while b₂ knockout mice show hypermotility. Two residues of the receptor located in the transmembrane segment M4 of b₂ confer 2-AG binding. With the aim to further describe this potential drug target we performed a cysteine scanning of the entire M4. All four residues in M4 affecting the stimulation here and the two already identified residues locate to the same side of the a-helix. Together with the fact that the important residues span > 18 Å, we conclude that the hydrophobic tail of the bound 2-AG molecule must be in a near linear conformation and that the site mainly locates to the inner leaflet but stretches into the outer leaflet of M4. The influence of the structure of the head group of the ligand molecule on the activity of the molecule was also investigated. In addition, we offer an explanation for the observation that stimulation by 2- AG is only observed at low agonist concentration and suggest an unusual mode of action of 2-AG. The identification of a functional binding site for 2-AG in the GABA_A receptor may have far-reaching consequences for the study of locomotion and sedation.