Several of the vaccinia virus core proteins are synthesized as large precursor proteins which are subsequently processed to smaller products during the course of viral maturation. Amino acid alignment of these proteins reveals a conserved Ala-Gly-X motif (AG*X) at their confirmed cleavage sites. To better understand the regulation of cleavage site selection, the sequence of the entire vaccinia virus genome was searched for the occurrence of this AG*X motif in predicted open reading frames. Of the 82 sites found, 19 resembled cleavage sites which have previously been shown to be actively processed, namely AG*A of P25K and P4b, and AG*S and AG*T of P4a. To test the universality of the AG*X motif utilization, immunological methods in concert with N-terminal microsequencing procedures have been used to determine which of the subset of predicted proteins containing AG*A sites are utilized in vivo. Of the seven AG*A-containing substrates, four were cleaved and three were not. Considering all the known AG*X processing events, it appears that only those proteins expressed at late times during infection and associated with the assembling virion are candidate substrates for proteolytic cleavage. Such proteins include P4a, P4b, P25K, and the newly identified P21K and P17K (derived from genes A17L and A12L, respectively). Although proteins such as DNA polymerase, P37K, and a host range protein contain a consensus cleavage site, they are excluded from processing. This proteolytic exclusion presumably occurs because these proteins do not meet both of the above criteria, which suggests that temporal expression or compartmentalization (substrate presentation) in the assembling virion may play a regulatory role in proteolysis.