Initiation of DNA replication from within the Epstein-Barr virus (EBV) latent cycle origin oriP occurs once per cell cycle and is almost entirely dependent upon cellular proteins. The human origin recognition complex (ORC) is recruited to oriP and orchestrates the events that lead to the initiation of replication. EBNA-1, the sole viral protein required for oriP-plasmid replication, binds four sites within the replicator but the role(s) it plays in the replication of oriP plasmids has not been elucidated. We investigated the recruitment of ORC to oriP in vivo and show that the binding of EBNA-1 to the replicator is necessary for the association of the ORC subunit Orc2 with the replicator. The minimal replicator of oriP consists of two EBNA-1 binding sites flanked by perfect 14-bp inverted repeats (a and b), but these repeats are dispensable for the association of Orc2 with the replicator. A mutational analysis of the 14-bp repeats provided additional support for a role for the telomere repeat binding protein 2 in oriP replicator function. We show that nucleotide differences between the oriP replicator of the B95-8 and Raji EBV genomes are not solely responsible for the inefficient utilization of this origin in the Raji EBV genome.