The proteasome catalyzes the majority of protein degradation in the cell and plays an integral role in cellular homeostasis. Control over proteolysis by the 20S core-particle (CP) proteasome is achieved by gated access of substrate; thus, an understanding of the molecular mechanism by which these gates regulate substrate entry is critical. We used methyl-transverse relaxation optimized nuclear magnetic resonance spectroscopy to show that the amino-terminal residues that compose the gates of the alpha subunits of the Thermoplasma acidophilum proteasome are highly dynamic over a broad spectrum of time scales and that gating termini are in conformations that extend either well inside (closed gate) or outside (open gate) of the antechamber. Interconversion between these conformers on a time scale of seconds leads to a dynamic regulation of 20S CP proteolysis activity.