DNA sequences which have structural features suggestive of their functioning as an origin of lytic-phase DNA replication were previously identified in both human herpesvirus 6B strain Z29 [HHV-6B (Z29)] and in HHV-6A (U1102). Plasmid constructs containing the putative HHV-6B (Z29) oriLyt element were replicated after transfection into permissive T cells, when trans-acting factors were provided by HHV-6B (R-1) infection. By using this assay, the HHV-6B (Z29) oriLyt was mapped to a minimal region of approximately 400 bp which lies upstream of the gene that is homologous to herpes simplex virus UL29, a region that carries an origin in other betaherpesviruses and in some alphaherpesviruses.