AMPA receptors are ligand-gated ion channels that show multiple conductance levels, indicating that gating of individual AMPA subunits is to some extent independent of the other subunits. To study AMPAR subunit interactions during activation gating, we recorded from single channels in the absence of channel block and desensitization and at negative and positive membrane potentials. In saturating glutamate, the relative occupancies of the various conductance levels are consistent with complete subunit independence. In contrast, the relative occupancies in subsaturating glutamate indicate that the channel switches between a low open probability mode and a high open probability mode in which the behavior of the channel is identical to that in saturating glutamate. These gating modes occur at both negative and positive potentials, with the high open probability mode becoming more prominent at positive potentials. The switch between gating modes and its modulation by voltage and other factors may constitute a novel mechanism regulating AMPAR-mediated synaptic activity.