The 3C-like proteinase (3CLpro) of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for anti-SARS drug discovery. It has been proposed and verified that the dimer was the active form of 3CLpro and only one protomer is active. In our previous work, we measured the dissociation constant (Kd) of the purified SARS 3CLpro using analytical ultracentrifugation at around 14.0 μmol·L-1. Using this Kd value, most of the SARS 3CLpro in the in vitro activity assay (1-3 μmol·L-1) might be in the monomer formand inactive. To explain this dilemma, we measured the enzyme activity change together with the enzyme concentration. By fitting the concentration dependent activity profile, the apparent dissociation constant was found to be 0.94 μmol·L-1, indicating a clear tendency toward substrate enhanced dimerization. This also explains why SARS 3CLpro was still active in the in vitro activity assay under a relatively low enzyme concentration. To further verify the substrate induced dimerization phenomenon, we selected a previously reported SARS 3CLpro isatin inhibitor, 1-(2-naphthlmethyl) isatin-5-carboxamide (5f), which has similar binding interactions with the substrate and we studied its influence on SARS 3CLpro dimer formation using analytical ultracentrifugation. 5f showed a strong ability to induce SARS 3CLpro dimer formation. By measuring the dimer and monomer distribution under different 5f concentrations, the EC50 of dimer induction was found to be about 1.0 μmol·L-1 under an enzyme concentration of 3.0 μmol·L-1. This implies that only one protomer in the SARS 3CLpro dimer binds to the inhibitor or the substrate. As the apparent association constant and thus the enzyme activity of SARS 3CLpro increases with the concentration of the substrate, this may be a smart way to allosterically regulate the hydrolysis of the SARS viral polyproteins and the correct assembly of virons.