There is a distinct possibility that general anesthetics exert their action on the postsynaptic receptor channels. The structural requirements for anesthetic binding in transmembrane channels, however, are largely unknown. High-resolution (1)H nuclear magnetic resonance and direct photoaffinity labeling were used in this study to characterize the volatile anesthetic binding sites in gramicidin A (gA) incorporated into sodium dodecyl sulfate (SDS) micelles and into dimyristoylphosphatidylcholine (DMPC) bilayers, respectively. To confirm that the structural arrangement of the peptide side chains can affect anesthetic binding, gA in nonchannel forms in methanol was also analyzed. The addition of volatile anesthetic halothane to gA in SDS with a channel conformation caused a concentration-dependent change in resonant frequencies of the indole amide protons of W9, W11, W13, and W15, with the most profound changes in W9. These frequency changes were observed only for gA carefully prepared to ensure a channel conformation and were absent for gA in methanol. For gA in DMPC bilayers, direct [(14)C]halothane photolabeling and microsequencing demonstrated dominant labeling of W9, less labeling of W11 and W13, and no significant labeling of W15. In methanol, gA showed much less labeling of any residues. Inspection of the 3-D structure of gA suggests that the spatial arrangements of the tryptophan residues in the channel form of gA, combined with the amphiphilic regions of lipid, create a favorable anesthetic binding motif.