We report the development of a method that should allow the insertion of a selective marker into any region of the Epstein-Barr virus (EBV) genome of strain B95-8 through homologous recombination with plasmids. In this method, EBV recombinants are isolated as G418-resistant, immortalized B-cell clones or as G418-resistant, latently infected subclones of Burkitt lymphoma cell lines. The presence of the productive replication origin of EBV, oriLyt, on the plasmid was found to increase the number of observed recombinant viruses by approximately 100-fold; this stimulation was observed when oriLyt was separated from the sites of recombination by several kilobases of nonhomologous DNA. Long segments of EBV DNA flanking the marker on the plasmid and/or a large plasmid size were inferred to be important for obtaining a high proportion of recombinant genomes that had recombined on both sides of the selective marker; otherwise, the recombinants that predominated had acquired the entire plasmid by recombining only on one side of the inserted marker. Therefore, to facilitate targeted insertion of genetic markers into the EBV genome, a cosmid vector carrying oriLyt was constructed and tested by using it to generate EBV mutants with the BALF2 open-reading frame disrupted.