Most general anesthetics including long chain aliphatic alcohols act as noncompetitive antagonists of the nicotinic acetylcholine receptor (nAChR). To locate the sites of interaction of a long chain alcohol with the Torpedo nAChR, we have used the photoactivatible alcohol 3-[(3)H]azioctanol, which inhibits the nAChR and photoincorporates into nAChR subunits. At 1 and 275 microm, 3-[(3)H]azioctanol photoincorporated into nAChR subunits with increased incorporation in the alpha-subunit in the desensitized state. The incorporation into the alpha-subunit was mapped to two large proteolytic fragments. One fragment of approximately 20 kDa (alpha V8-20), containing the M1, M2, and M3 transmembrane segments, showed enhanced incorporation in the presence of agonist whereas the other of approximately 10 kDa (alpha V8-10), containing the M4 transmembrane segment, did not show agonist-induced incorporation of label. Within alpha V8-20, the primary site of incorporation was alpha Glu-262 at the C-terminal end of alpha M2, labeled preferentially in the desensitized state. The incorporation at alpha Glu-262 approached saturation between 1 microm, with approximately 6% labeled, and 275 microm, with approximately 30% labeled. Low level incorporation was seen in residues at the agonist binding site and the protein-lipid interface at approximately 1% of the levels in alpha Glu-262. Therefore, the primary binding site of 3-azioctanol is within the ion channel with additional lower affinity interactions within the agonist binding site and at the protein-lipid interface.