The cis-acting signals required for cleavage and encapsidation of the herpes simplex virus type 1 genome lie within the terminally redundant region or a sequence. The a sequence is flanked by short direct repeats (DR1) containing the site of cleavage, and quasi-unique regions, Uc and Ub, occupy positions adjacent to the genomic L and S termini, respectively, such that a novel fragment, Uc-DR1-Ub, is generated upon ligation of the genomic ends. The Uc-DR1-Ub fragment can function as a minimal packaging signal, and motifs have been identified within Uc and Ub that are conserved near the ends of other herpesvirus genomes (pac2 and pac1, respectively). We have introduced deletion and substitution mutations within the pac regions of the Uc-DR1-Ub fragment and assessed their effects on DNA packaging in an amplicon-based transient transfection assay. Within pac2, mutations affecting the T tract had the greatest inhibitory effect, but deletion of sequences on either side of this element also reduced packaging, suggesting that its position relative to other sequences within the Uc-DR1-Ub fragment is likely to be important. No single region essential for DNA packaging was detected within pac1. However, mutants lacking the G tracts on either side of the pac1 T-rich motif exhibited a reduced efficiency of serial propagation, and alteration of the sequences between DR1 and the pac1 T element also resulted in defective generation of Ub-containing terminal fragments. The data are consistent with a model in which initiation and termination of packaging are specified by sequences within Uc and Ub, respectively.