Electron paramagnetic resonance (EPR) spectroscopy is a powerful technique that permits the study of membrane-embedded proteins in its lipid environment by assessing the interaction of spin labels with the protein in its natural environment (i.e., native membranes) or in reconstituted systems prepared with exogenous lipid species. Nicotinic acetylcholine receptors (AChRs) contain a large surface in intimate contact with the lipid membrane. AChRs, members of the Cys-loop receptor superfamily, have essential functional roles in the nervous system and its malfunctioning has been considered as the origin of several neurological diseases including Alzheimer's disease, drug addiction, depression, and schizophrenia. In this regard, these receptors have been extensively studied as therapeutic targets for the action of several drugs. The majority of the marketed medications bind to the neurotransmitter sites, the so-called agonists. However, several drugs, some of them still in clinical trials, interact with non-competitive antagonist (NCA) binding sites. A potential location for these binding sites is the proper ion channel, blocking ion flux and thus, inhibiting membrane depolarization. However, several NCAs also bind to the lipid-protein interface, modulating the AChR functional properties. The best known examples of these NCAs are local and general anesthetics. Several endogenous molecules such as free fatty acids and neurosteroids also bind to the lipid-protein interface, probably mediating important physiological functions. Phospholipids, natural components of lipid membranes interacting with the AChR, are also essential to maintain the structural and functional properties of the AChR. EPR studies showed that local anesthetics bind to the lipid-protein interface by essentially the same dynamic mechanisms found in lipids, and that local and general anesthetics preferably decrease the phospholipid but not the fatty acid interactions with the AChR. This is consistent with the existence of annular and non-annular lipid domains on the AChR.