Vaccinia virus relies on a series of proteolytic cleavage events involving two viral proteins, I7 and G1, to complete its life cycle. Furthermore, G1 itself is cleaved during vaccinia virus infection. However, convincing evidence is lacking to show whether G1 participates in autoproteolysis or is a substrate of another protease. We employed both biochemical and cell-based approaches to investigate G1 cleavage. G1, when expressed in bacteria, rabbit reticulocyte lysates or HeLa cells, was not processed. Moreover, G1 was cleaved in infected cells, but only in the presence of virus late gene expression; cleavage was strongly inhibited by proteasome inhibitors. Thus, these results imply a more complex G1 cleavage reaction than previously envisaged.