Autocleavage assay and peptide-based cleavage assay were used to study the substratespecificity of 3CL protease from the severe acute respiratory syndrome coronavirus.It was found that therecognition between the enzyme and its substrates involved many positions in the substrate,at least includingresidues from P4 to P2′.The deletion of either P4 or P2′residue in the substrate would decrease its cleavageefficiency dramatically.In contrast to the previous suggestion that only small residues in substrate could beaccommodated to the S1′subsite,we have found that bulky residues such as Tyr and Trp were also acceptable.In addition,based on both peptide-based assay and autocleavage assay,Ile at the P1′position could not behydrolyzed,but the mutant L27A could hydrolyze the Ile peptide fragment.It suggested that there was astereo hindrance between the S1′subsite and the side chain of Ile in the substrate.All 20 amino acids exceptPro could be the residue at the P2′position in the substrate,but the cleavage efficiencies were clearlydifferent.The specificity information of the enzyme is helpful for potent anti-virus inhibitor design and usefulfor other coronavirus studies. Autocleavage assay and peptide-based cleavage assay were used to study the substratespecific of 3CL protease from the severe acute respiratory syndrome coronavirus. It was found that there isognognopathy between the enzyme and its substrates involved many positions in the substrate, at least includingores from P4 to P2 ’. The deletion of either P4 or P2’residue in the substrate would decrease its cleavageefficiency.In.In contrast to the previous suggestion that only small residues in substrate could be accommodated to the S1’subsite, we have found that bulky residues such as Tyr and Trp were also acceptable. In addition, based on both peptide-based assay and autocleavage assay, Ile at the P1’position could not be hydrolyzed, but the mutant L27A could hydrolyze the peptide fragment. It suggested that there was astereo hindrance between the S1 ’Subsite and the side chain of Ile in the substrate. A11 20 amino acids exceptPro could be the residue at the P2’position in the subs trate, but the cleavage of nutrients are clearly different. the specificity information of the enzyme is helpful for potent anti-virus inhibitor design and useful for other coronavirus studies.