Protein degradation plays a critical role in cellular homeostasis, in regulating the cell cycle, and in the generation of peptides that are used in the immune response. The 20S proteasome core particle (CP), a barrel-like structure consisting of four heptameric protein rings stacked axially on top of each other, is central to this process. CP function is controlled by activator complexes that bind 75 Å away from sites catalyzing proteolysis, and biochemical data are consistent with an allosteric mechanism by which binding is communicated to distal active sites. However, little structural evidence has emerged from the high-resolution images of the CP. Using methyl TROSY NMR spectroscopy, we demonstrate that in solution, the CP interconverts between multiple conformations whose relative populations are shifted on binding of the 11S activator or mutation of residues that contact activators. These conformers differ in contiguous regions of structure that connect activator binding to the CP active sites, and changes in their populations lead to differences in substrate proteolysis patterns. Moreover, various active site modifications result in conformational changes to the activator binding site by modulating the relative populations of these same CP conformers. This distribution is also affected by the binding of a small-molecule allosteric inhibitor of proteolysis, chloroquine, suggesting an important avenue in the development of therapeutics for proteasome inhibition.