Neuronal nicotinic acetylcholine receptors (nAChRs) are potential targets for a wide variety of general anesthetics. We recently showed that alpha (4)beta (2) nAChRS are more sensitive than alpha (4)beta (4) receptors to the gaseous anesthetics nitrous oxide and xenon. The present study examines chimeric and point mutant rat nAChRs expressed in Xenopus oocytes and identifies a single amino acid residue (beta (2)-Val(253) or beta (4)-Phe(255)) near the middle of the second transmembrane segment (TM2) that determines gaseous anesthetic sensitivity. Mutations of this residue in beta subunits and the homologous residue of alpha (4) subunits (alpha (4)-Val(254)) showed that this position also determines sensitivities of nAChRs to acetylcholine, isoflurane, pentobarbital, and hexanol, In contrast, these mutations did not affect actions of ketamine, The positively charged sulfhydryl-specific reagent methanethiosulfonate ethylammonium reacted with a cysteine introduced at alpha (4)-Val(254) or beta (2)-Val(258), and irreversibly reduced anesthetic sensitivities of nAChRs, Propyl methanethiosulfonate is an anesthetic analog that covalently binds to a TM2 site of gamma -aminobutyric acid, and glycine receptors and irreversibly enhances receptor function. However, propyl methanethiosulfonate reversibly inhibited cysteine-substitution mutants at alpha (4)-Val(254) or beta (2)-Val(253) of nAChRs, and did not affect anesthetic sensitivity. Thus, residues alpha (4)-Val(254) and beta (2)-Val(253) alter channel gating and determine anesthetic sensitivity of nAChRs, but are not likely to be anesthetic-binding sites.