The vaccinia virus I7L gene product is predicted to be a cysteine proteinase and is demonstrated in this study to be responsible for cleavage of each of the three major core protein precursors (P4a, P4b, and P25K) in vivo. Mutagenesis of the putative catalytic triad of I7L or of the cleavage sites in the core protein precursors inhibits processing. A truncated protein lost the ability to cleave the core protein precursors.