The endocannabinoid, N-arachidonoylethanolamine (anandamide; AEA) is known to interact with voltage-gated K ϩ (Kv) channels in a cannabinoid receptor-independent manner. AEA modulates the functional properties of Kv channels, converting channels with slowly inactivating current into apparent fast inactivation. In this study, we characterize the mechanism of action and binding site for AEA on Kv1.5 channels expressed on HEK-293 cells using the patch-clamp techniques. AEA exhibited high-potency block (IC 50 Ϸ 200 nM) from the cytoplasmic membrane surface, consistent with open-channel block. Alanine-scanning mutagenesis revealed that AEA interacts with two crucial ␤-branching amino acids, Val505 and Ile508 within the S6 domain. Both residues face toward the central cavity and constitute a motif that forms a hydrophobic ring around the ion conduction pathway. This hydrophobic ring motif may be a critical determinant of cannabinoid receptor-independent AEA modulation in other K ϩ channel families.