The vaccinia virus G1L open-reading frame is predicted to be a metalloproteinase based upon the presence of a conserved zinc-binding motif. Western blot analysis demonstrates G1L undergoes proteolytic processing during the course of infection, although the significance of this event is unknown. In order to determine which amino acid residues are important for G1L activity, a plasmid-borne library of G1L constructs containing mutations in and about the active site was created. Transient expression analysis coupled with a trans complementation assay of a conditionally-lethal mutant virus suggest that, of the mutants, only glutamic acid 120 is non-essential for G1L processing to occur.