The 20S proteasome core particle is a molecular machine that plays a central role in the regulation of cellular function through proteolysis, and it has emerged as a valuable drug target for certain classes of cancers. Central to the development of new and potent pharmaceuticals is an understanding of the mechanism by which the proteasome cleaves substrates. A number of high-resolution structures of the 20S proteasome with and without inhibitors have emerged that provide insight into the chemistry of peptide bond cleavage and establish the role of Thr1 Oγ1 as the catalytic nucleophile. The source of the base that accepts the Thr1 Hγ1 is less clear. Using a highly deuterated sample of the proteasome labeled with (13)CH3 at the Thr-γ positions, the pKA of the Thr1 amino group has been measured to be 6.3 and hence deprotonated in the range of maximal enzyme activity. This provides strong evidence that the terminal amino group of Thr1 serves as the base in the first step of the peptide bond cleavage reaction.