An assay has been developed and used to locate an origin of DNA replication on the herpes simplex virus type 1 (HSV-1) genome. Baby hamster kidney cells were transfected with circular plasmid molecules containing cloned copies of HSV-1 DNA fragments, and helper functions were provided by superinfection with wild-type HSV-1. The presence of an HSV-1 origin of replication within a plasmid enabled amplification of the vector DNA sequences, which was detected by the incorporation of [32P]orthophosphate. By screening various HSV-1 DNA fragments it was possible to identify a 995-bp fragment that maps entirely within the reiterated sequences flanking the short unique region of the viral genome and contains all the cis-acting signals necessary to function as an origin of viral DNA replication. The products of plasmid replication were shown to be high mol. wt. DNA molecules consisting of tandem duplications of the complete plasmid, suggesting that replication was occurring by a rolling-circle mechanism.