Vaccinia virus (VV), the best-studied member of the Poxviridae, is a complex enveloped virus that replicates in the cytoplasm of infected cells. Its linear double-stranded genome is approximately 190 kbp and encodes nearly 200 proteins including those involved in viral DNA replication, transcription, and assembly of virions. Viral morphogenesis is a complex process and distinct types of virions that have different roles in the virus life cycle are produced. The transition from the immature virus (IV) stage to the intracellular mature virus (IMV) stage occurs through a number of events that are not well understood. During this transition, certain core and membrane proteins undergo proteolytic processing at consensus AG/X sites. The protease(s) involved in these cleavages was not known, however two virus-encoded proteins, I7 and G1 were the most likely candidates. In order to investigate the role of these two proteins in the proteolytic maturation of viral structural proteins, two conditional-lethal mutant viruses were constructed in which the expression of the I7L or G1L genes was individually placed under the control of an Escherichia coli lac operon. Evidence was found that I7 and G1 are vaccinia virus encoded proteases, I7 a cysteine protease and G1 a metalloprotease. This study showed that I7, but not G1, is required for the AG/X-specific cleavages of viral membrane and core proteins. The substrate of G1 is not known yet, but the protein itself was found in a cleaved form associated with purified particles. Furthermore, both proteins were shown to have a role in morphogenesis and to be essential for virus infectivity.