The coronavirus, mouse hepatitis virus strain A59 (MHV), expresses a chymotrypsin-like cysteine proteinase (3CLpro) within the gene 1 polyprotein. The MHV 3CLpro is similar to the picornavirus 3C proteinases in the relative location of confirmed catalytic histidine and cysteine residues and in the predicted use of Q/(S, A, G) dipeptide cleavage sites. However, less is known concerning the participation of aspartic acid or glutamic acid residues in catalysis by the coronavirus 3C-like proteinases or of the precise coding sequence of 3CLpro within the gene 1 polyprotein. In this study, aspartic acid residues in MHV 3CLpro were mutated and the mutant proteinases were tested for activity in an in vitro trans cleavage assay. MHV 3CLpro was not inactivated by substitutions at Asp3386 (D53) or Asp3398 (D65), demonstrating that they were not catalytic residues. MHV 3CLpro was able to cleave at a glutamine-glycine (QG3607-8) dipeptide within the 3CLpro domain upstream from the predicted carboxy-terminal QS3636-6 cleavage site of 3CLpro. The predicted full-length 3CLpro (S3334 to Q3635) had an apparent mass of 27 kDa, identical to the p27 3CLpro in cells, whereas the truncated proteinase (S3334 to Q3607) had an apparent mass of 24 kDa. This 28-amino-acid carboxy-terminal truncation of 3CLpro rendered it inactive in a trans cleavage assay. Thus, MHV 3CLpro was able to cleave at a site within the putative full-length proteinase, but the entire predicted 3CLpro domain was required for activity. These studies suggest that the coronavirus 3CL-proteinases may have a substantially different structure and catalytic mechanism that other 3C-like proteinases.