1. Single channel currents were recorded from HEK 293 cells expressing recombinant mouse adult (α2βδε) and embryonic (α2βδγ) acetylcholine receptors (AChRs) containing a mutation at residue D200 of the α-subunit. Rate and equilibrium constants for AChR activation were estimated from open and closed times obtained over a range of ACh concentrations. 2. Mutation of αD200 to asparagine (αD200N) dramatically slows the rate constant of channel opening, with adult AChRs slowing 100-fold and embryonic AChRs slowing 400-fold. The rate constant of channel closing increases 3-fold, resulting in a decrease of the gating equilibrium constant of up to 1200-fold. In contrast to channel gating steps, ACh-binding steps are only modestly effected by αD200N. 3. Introduction of a potential glycosylation site in αD200N cannot account for the effect on channel gating because eliminating the consensus for glycosylation with the mutation αD200N + T202V fails to restore efficient gating. Gating is similarly impaired with the substitutions of E, K and Q at position α200. 4. The agonists carbamylcholine and tetramethylammonium also activate the αD200N AChR, but with channel opening rates even slower than with ACh. The agonist dependence of the opening rate constant is similar in αD200N and wild type AChRs. 5. AChRs containing D200N at just one of the two α-subunits show either small or large changes in the gating equilibrium constant, presumably due to the presence of the mutation at either the αδ or αε/αγ sites. The changes in free energy of channel gating show that the contribution of each binding site is nearly independent. However, the sites do not contribute equally to gating, as an αD200N mutation at the αε or αγ binding site slows channel opening relatively more than at the αδ site.