The 3C-like proteinase (3CLpro) of mouse hepatitis virus (MHV) is predicted to cleave at least 11 sites in the 803-kDa gene 1 polyprotein, resulting in maturation of proteinase, polymerase, and helicase proteins. However, most of these cleavage sites have not been experimentally confirmed and the proteins have not been identified in vitro or in virus-infected cells. We used specific antibodies to identify and characterize a 22-kDa protein (p1a-22) expressed from gene 1 in MHV A59-infected DBT cells. Processing of p1a-22 from the polyprotein began immediately after translation, but some processing continued for several hours. Amino-terminal sequencing of p1a-22 purified from MHV-infected cells showed that it was cleaved at a putative 3CLpro cleavage site, Gln_Ser4014 (where the underscore indicates the site of cleavage), that is located between the 3CLpro domain and the end of open reading frame (ORF) 1a. Subclones of this region of gene 1 were used to express polypeptides in vitro that contained one or more 3CLpro cleavage sites, and cleavage of these substrates by recombinant 3CLpro in vitro confirmed that amino-terminal cleavage of p1a-22 occurred at Gln_Ser4014. We demonstrated that the carboxy-terminal cleavage of the p1a-22 protein occurred at Gln_Asn4208, a sequence that had not been predicted as a site for cleavage by MHV 3CLpro. Our results demonstrate the usefulness of recombinant MHV 3CLpro in identifying and confirming cleavage sites within the gene 1 polyprotein. Based on our results, we predict that at least seven mature proteins are processed from the ORF 1a polyprotein by 3CLpro and suggest that additional noncanonical cleavage sites may be used by 3CLpro during processing of the gene 1 polyprotein.