Previous studies have suggested that cleavage of vaccinia virus core protein precursors occurs within the consensus tripeptide motif -A-G decreases X-. As an approach to delineate the sequence and structural features of the precursor polypeptides that are responsible for directing site-specific scission within this element, site-directed mutagenesis procedures were employed in concert with an in vivo trans-processing assay of the P25K: FLAG reporter plasmid. The results obtained suggest that residue occupancy at the P1' site (following the nomenclature of Schechter and Berger (Schechter, I., and Berger, A. (1976) Biochem. Biophys. Res. Commun. 27, 157-162), the positions at the amino- and carboxyl-proximal residues are indicated as P1, P2, etc., and P1', P2', etc., respectively) was extremely permissive, with only a proline substitution blocking cleavage. In contrast, the permissible occupancy of the P1 (serine or alanine) and P2 (cysteine, serine, or asparagine) sites was extremely restricted. Analysis of P1/P2 double mutants supported this conclusion and suggested additional levels of combinatorial stringency. Insertion or deletion of sequences immediately adjacent (amino- or carboxyl-terminal) to the -A-G-X- motif completely abrogated cleavage, suggesting the presence of additional important structural determinants. Mutation of the conserved proline or basic amino acid residues in these regions had no effect on cleavage, whereas it appeared that the presence of a hydrophobic residue in the P4 site was required.