The cation-conducting channel of the nicotinic acetylcholine (ACh) receptor is lined by the first (M1) and second (M2) membrane-spanning segments of each of its five subunits. Six consecutive residues, alphaS239 to alphaT244, in the alpha subunit M1-M2 loop and at the intracellular end of M2 were mutated to cysteine. The accessibility of the substituted cysteines were probed with small, cationic, sulfhydryl-specific reagents added extracellularly and intracellularly. In the closed state of the channel, there is a barrier to these reagents added from either side between alphaG240 and alphaT244. ACh induces the removal of this barrier, which acts as an activation gate. The residues alphaG240, alphaE241, alphaK242, and alphaT244 line a narrow part of the channel, in which this gate is located.