The interaction of general anesthetics at the lipid/protein interface of the nicotinic acetylcholine receptor reconstituted in dioleoylphosphatidylcholine bilayers at various lipid/protein ratios has been studied using the electron spin resonance spectra of phosphatidylcholine spin-labeled at the fourteenth acyl carbon (14-PCSL). In addition to the bilayer spectrum, the spin label reported a more motionally restricted environment whose contribution increased with increasing protein/lipid ratio. Exchange between these two environments occurred at a rate of approx. 6 x 10(7) s-1. The motionally restricted, protein-associated 14-PCSL had a rotational correlation time of about 10-20 ns, an order of magnitude slower than when in the bilayer. Addition of 1-hexanol (up to 16 mM) to the reconstituted receptor perturbed the acyl chains of the bulk lipid phase, but the motional properties of the lipid acyl chains at the protein/lipid interface near the membrane center were not significantly perturbed on the EPR motional time-scale. Similarly, anesthetics that were less effective at perturbing the bilayer, such as pentobarbital (up to 2 mM) and isoflurane (7 mM), did not perturb the lipid/protein interface on the conventional EPR motional time scale.