We have examined changes in single-channel behavior that result from conservative amino acid substitutions at the Cys230 residue in the putative first transmembrane region (M1) of the murine nicotinic acetylcholine receptor. Mutations made in the gamma subunit altered the energy barrier for a single closing rate constant in proportion to the size of the substituted side chain. One of these substitutions, when made in the alpha subunits, had no effect on gating. No mutations altered permeation. We conclude that the region surrounding the M1 Cys is involved in the gating of the nicotinic acetylcholine receptor and that the gamma subunit contributes significantly to the control of channel closure.