DNA replication from oriP of Epstein-Barr virus is mediated by the virus replication factor EBNA1 and cellular factors and occurs approximately once in each cell cycle. We have identified a minimal oriP element that is necessary and sufficient for DNA replication. We transfected plasmids containing several oriP fragments into HeLa cells expressing EBNA1 and analyzed their replication during four days after transfection using the methylation sensitive restriction endonuclease DpnI. All the oriP fragments containing the four EBNA1-binding sites known as the dyad symmetry sequence (DS) initiated DNA replication. The sequences flanking DS were not essential for DNA replication, but deletion of two or three EBNA1-binding sites in DS significantly reduced or totally abolished its replication activity. These results indicated that the four EBNA1-binding sites in DS constitute the minimal oriP element for DNA replication and suggest that DNA replication is initiated by recruitment of cellular replication factors onto or near the minimal oriP by EBNA1. We also found that the minimal oriP initiated DNA replication in mouse fibroblasts expressing EBNA1 but worked only at reduced efficiency, suggesting species specificity in DNA replication machineries.