The 26S proteasome is a 2500 kDa protease complex that degrades polyubiquitylated proteins by a mechanism that requires ATP hydrolysis. It also degrades short non-ubiquitylated peptides and certain unstructured proteins by an energy-independent mechanism that requires bound ATP to maintain its component subcomplexes, the 20S proteasome and PA700, in a functionally assembled state. Proteolysis of both types of substrate requires PA700-induced opening of reversible gates at substrate-access pores of the 20S proteasome. In the present study we demonstrate that the rate of peptide substrate hydrolysis, a functional monitor of gate opening, is regulated variably by multiple effectors. ATPgammaS (adenosine 5'-[gamma-thio]triphosphate) and other non-hydrolysable ATP analogues increased peptide substrate hydrolysis by intact 26S proteasomes. Thus nucleotides that maintained 26S proteasome structure, but did not support ATP hydrolysis or the degradation of polyubiquitylated proteins, promoted enhanced rates of peptide hydrolysis. Polyubiquitin and a peptoid that binds selectively to a single ATPase subunit of PA700 also increased rates of peptide hydrolysis but had disparate effects on rates of ATP hydrolysis. The effect of polyubiquitin was specific for ubiquitin-ubiquitin linkages that supported proteolysis of protein substrates. These results indicate that gating of the 26S proteasome is not a simple two-state process but can be variably modulated. Our results suggest that modulated gating of the proteasome may be an important element of the mechanism of proteolysis of polyubiquitylated proteins.