We have identified a cis-acting element of Epstein-Barr virus (EBV) that mediates viral DNA replication during the lytic phase of this virus's life cycle. This lytic origin of DNA replication, termed oriLyt, is complex in structure in that it contains multiple regions that are required for replication and additional DNA sequences that increase replication. One of the required regions of oriLyt can be functionally substituted by a transcriptional enhancing element. DNA replication mediated by oriLyt depends on EBV DNA polymerase and yields a concatemeric molecule. A vector, which contains both oriP (the EBV plasmid origin of replication) and oriLyt, can be maintained as a plasmid in latently EBV-infected cells and can be amplified 100- to 1000-fold in cells in which the lytic phase of the viral life cycle is induced.