Vaccinia virus maturation into infectious particles appears to be dependent on the proteolytic processing of at least five viral proteins, each containing a conserved AG*X cleavage motif and each requiring proper association with the previrion particle. To identify the responsible proteinase, a transcriptionally controlled trans-processing assay was developed to monitor cleavage at the permissive AG*S site of the P25K core protein precursor. This assay led to the putative identification of a VV proteinase encoded by open reading frame G1L. The predicted protein contains an HXXEH sequence which is a direct inversion of the active site consensus sequence present in thermolysin and other metalloendopeptidases. Site-directed mutation of this consensus sequence suggests that the G1L protein may be a novel, virus-encoded metalloendoproteinase, although confirmation of this activity must await the development of a suitable cell-free processing assay.