Gene 1 of the murine coronavirus, MHV-A59, encodes approximately 800 kDa of protein products within two overlapping open reading frames (ORFs 1a and 1b). The gene is expressed as a polyprotein that is processed into individual proteins, presumably by virus-encoded proteinases. ORF 1a has been predicted to encode proteins with similarity to viral and cellular proteinases, such as papain, and to the 3C proteinases of the picornaviruses (A. E. Gorbalenya, A. P. Donchenko, V. M. Blinov, and E. V. Koonin, FEBS Lett. 243:103-114, 1989; A. E. Gorbalenya, E. V. Koonin, A. P. Donchenko, and V. M. Blinov, Nucleic Acids Res. 17:4847-4861, 1989). We have cloned into a T7 transcription vector a cDNA fragment containing the putative 3C-like proteinase domain of MHV-A59, along with portions of the flanking hydrophobic domains. The construct was used to express a polypeptide in a combined in vitro transcription-translation system. Major polypeptides with molecular masses of 38 and 33 kDa were detected at early times, whereas polypeptides with molecular masses of 32 and 27 kDa were predominant after 30 to 45 min and appeared to be products of specific proteolysis of larger precursors. Mutations at the putative catalytic histidine and cysteine residues abolished the processing of the 27-kDa protein. Translation products of the pGpro construct were able to cleave the 27-kDa protein in trans from polypeptides expressed from the noncleaving histidine or cysteine mutants. The amino-terminal cleavage of the 27-kDa protein occurred at a glutamine-serine dipeptide as previously predicted. This study provides experimental confirmation that the coronaviruses express an active proteinase within the 3C-like proteinase domain of gene 1 ORF 1a and that this proteinase utilizes at least one canonical QS dipeptide as a cleavage site in vitro.