Herpesviruses have large double-stranded linear DNA genomes that are formed by site-specific cleavage from complex concatemeric intermediates. In this process, only one of the two genomic ends are formed on the concatemer. Although the mechanism underlying this asymmetry is not known, one explanation is that single genomes are cleaved off of concatemer ends in a preferred direction. This implies that cis elements control the direction of packaging. Two highly conserved cis elements named pac1 and pac2 lie near opposite ends of herpesvirus genomes and are important for cleavage and packaging. By comparison of published reports and by analysis of two additional herpesviruses, we found that pac2 elements lie near the ends formed on replicative concatemers of four herpesviruses: herpes simplex virus type 1, equine herpesvirus 1, guinea pig cytomegalovirus, and murine cytomegalovirus. Formation of pac2 ends on concatemers depended on terminal cis sequences, since ectopic cleavage sites engineered into the murine cytomegalovirus genome mediated formation of pac2 ends on concatemers regardless of the orientation of their insertion. These findings are consistent with a model in which pac2 elements at concatemer ends impart a directionality to concatemer packaging by binding proteins that initiate insertion of concatemer ends into empty capsids.Herpesviruses have large (130 to 235 kb) linear doublestranded DNA genomes that replicate via concatemeric intermediates consisting of head-to-tail linked genomes (1,3,17,22,23,31,32,41). The concatemers are packaged into preformed capsids and cleaved at precise locations to release unit length genomes within the capsids (31). In previous studies, we analyzed human cytomegalovirus (HCMV) concatemeric DNA for the presence of termini similar to those found on genomic DNA (23). The HCMV genome contains long and short components, or arms, that consist of unique regions flanked by inverted repeats (30). The ends of the genome are therefore referred to as the long arm end and the short arm end. We observed that HCMV concatemers contain terminal restriction fragments from the short arm end of the genome but lack terminal fragments from the long arm end (23). Similar findings of one but not both genomic termini on concatemeric DNA have since been reported for herpes simplex virus type 1 (HSV-1) (22, 32, 41) and equine herpesvirus 1 (EHV-1) (33), suggesting that this may be a characteristic of all herpesviruses. To explain our observation for HCMV, we proposed a model in which short arm termini on concatemer ends are inserted into empty capsids and packaged until full genome lengths have entered and cleavage sites are encountered. Cleavage then releases unit genomes into the capsids, generating long arm termini on the newly formed genomes and short arm termini on the newly formed concatemer ends. The short arm termini on concatemer ends are then free to encounter additional empty capsids and reinitiate the packaging process (23).The directionality proposed by this model suggests that cisac...